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Root rot of Panax notoginseng, a precious Chinese medicinal plant, seriously impacts its sustainable production. However, the molecular regulatory mechanisms employed by P. notoginseng against root rot pathogens, including Fusarium solani, are still unclear. In this study, the PnMYB2 gene was isolated, and its expression was affected by independent treatments with four signaling molecules (methyl jasmonate, ethephon, salicylic acid, and hydrogen peroxide) as assessed by quantitative real-time PCR. Moreover, the PnMYB2 expression level was induced by F. solani infection. The PnMYB2 protein localized to the nucleus and may function as a transcription factor. When overexpressed in transgenic tobacco, the PnMYB2 gene conferred resistance to F. solani. Jasmonic acid (JA) metabolism and disease resistance-related genes were induced in the transgenic tobacco, and the JA content significantly increased compared with in the wild type. Additionally, transcriptome sequencing, Kyoto Encyclopedia of Genes and Genomes annotation enrichment, and metabolic pathway analyses of the differentially expressed genes in the transgenic tobacco revealed that JA metabolic, photosynthetic, and defense response-related pathways were activated. In summary, PnMYB2 is an important transcription factor in the defense responses of P. notoginseng against root rot pathogens that acts by regulating JA signaling, photosynthesis, and disease-resistance genes.

Introduction Transcription factors (TFs) and cis-regulatory elements (CREs) control gene transcripts involved in various biological processes. We hypothesize that TFs and CREs can be effective molecular tools for De Novo regulation designs to engineer plants. Objectives We selected two Arabidopsis TF types and two tobacco CRE types to design a De Novo regulation and evaluated its effectiveness in plant engineering. Methods G-box and MYB recognition elements (MREs) were identified in four Nicotiana tabacum JAZs (NtJAZs) promoters. MRE-like and G-box like elements were identified in one nicotine pathway gene promoter. TF screening led to select Arabidopsis Production of Anthocyanin Pigment 1 (PAP1/MYB) and Transparent Testa 8 (TT8/bHLH). Two NtJAZ and two nicotine pathway gene promoters were cloned from commercial Narrow Leaf Madole (NL) and KY171 (KY) tobacco cultivars. Electrophoretic mobility shift assay (EMSA), cross-linked chromatin immunoprecipitation (ChIP), and dual-luciferase assays were performed to test the promoter binding and activation by PAP1 (P), TT8 (T), PAP1/TT8 together, and the PAP1/TT8/Transparent Testa Glabra 1 (TTG1) complex. A DNA cassette was designed and then synthesized for stacking and expressing PAP1 and TT8 together. Three years of field trials were performed by following industrial and GMO protocols. Gene expression and metabolic profiling were completed to characterize plant secondary metabolism. Results PAP1, TT8, PAP1/TT8, and the PAP1/TT8/TTG1 complex bound to and activated NtJAZ promoters but did not bind to nicotine pathway gene promoters. The engineered red P + T plants significantly upregulated four NtJAZs but downregulated the tobacco alkaloid biosynthesis. Field trials showed significant reduction of five tobacco alkaloids and four carcinogenic tobacco specific nitrosamines in most or all cured leaves of engineered P + T and PAP1 genotypes. Conclusion G-boxes, MREs, and two TF types are appropriate molecular tools for a De Novo regulation design to create a novel distant-pathway cross regulation for altering plant secondary metabolism.

BACKGROUND: R2R3-MYB transcription factor (TF) family plays important roles in various biological processes in many plants, especially in the regulation of plant flavonoid accumulation. The fruit of Lonicera caerulea contains abundant anthocyanin. OBJECTIVE: The R2R3-MYB TF family was systematically analyzed according to the RNA-seq data, and the R2R3-MYB candidate genes that were involved in anthocyanin biosynthesis in the fruit of Lonicera caerulea were screened. METHODS: The R2R3-MYB TFs in Lonicera caerulea were identified, and the physical and chemical properties, protein conserved sequence alignment and motifs of each R2R3-MYB TFs were analyzed using bioinformatics methods. The expression levels of these genes and anthocyanin levels in different tissues and different developmental stages of fruit were determined by RT-qPCR and pH shift method. RESULTS: A total of 59 genes encoding R2R3-MYB TFs in Lonicera caerulea were identified and clustered into 20 subgroups (C1 to C20) based on the relationship to AtR2R3-MYBs. Expression profiles showed that the expression of CL6086 and CL552 in fruit were higher than other tissues, and upregulated in the veraison fruit compared to the green ripe fruit. As the expression of the two genes was concurrent with the anthocyanin content, and showed high correlation with anthocyanin biosynthetic structural genes, they were considered as closely related to anthocyanin biosynthesis in the fruit. CONCLUSION: The results provide a systematic analysis of LcR2R3-MYBs, and the foundation for further molecular mechanisms research of anthocyanin biosynthesis regulated by R2R3-MYB in the fruit of Lonicera caerulea.

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.

Plants, in nature are challenged by various environmental stresses (both biotic and abiotic), which distort the plant growth, development and crop yield. A wide range of insect herbivores attack plants across the globe, throughout the year, causing huge loss to the productivity of crop species. Being sessile, the plants respond to the crisis using a wide range of defense responsive elements which at the transcriptional level, includes the transcription factors, like WRKY, NAC, AP2/ERF, MYB, MYC, bZIP, bHLH and others. Increasing evidences suggest a pivotal role of WRKY TFs in the regulation of plant defense response against herbivory in Arabidopsis and other important crop plants. Unlike pathogenic attack, very limited information is available on the role of different WRKYs in herbivore-plant interaction. In this review, we will thus majorly focus on the role of WRKY transcription factors and its regulation in response to herbivore attack while summarizing the recent developments from the field of transcriptional, post-transcriptional and epigenetic regulation of WRKYs in response to herbivory attack with the prospects for future implementation in research.

Water deficit inhibits maize (Zea mays L.) seedling growth and yield. Application of exogenous melatonin can improve drought tolerance of corn, but little is known regarding the transcriptional mechanisms of melatonin-mediated drought tolerance in maize. Increased understanding of the effects of melatonin on maize plants under drought stress is vital to alleviate the adverse effects of drought on food production in the future. The aim of this investigation was to use physiological and transcriptome analyses for exploring the possible mechanisms of exogenous melatonin against drought stress in maize. In this study, maize seedlings were subjected to drought stress and some were treated with exogenous melatonin. The physiological results showed that melatonin inhibited H2O2 accumulation and promoted the scavenging of excessive reactive oxygen species to reduce oxidative damage in maize leaves. Transcriptomic analysis identified 957 differentially expressed genes between melatonin and non-melatonin treatment groups. Further detailed analyses suggested that melatonin-regulated genes are mainly related to glutathione metabolism, calcium signaling transduction, and jasmonic acid biosynthesis. Some transcription factor families, such as WRKY, AP2/ERF-ERF, MYB, NAC, and bZIP, were also activated by exogenous melatonin. Moreover, crosstalk between melatonin and other hormones that mediate drought tolerance was observed. In conclusion, the combination of physiological and transcriptome analyses revealed some mechanisms underlying the role of melatonin in alleviating drought; knowledge of these mechanisms may assist in successful maize cultivation under drought stress.

Senescence of leaves is initiated by differential expression of genes of transcription factors and is controlled by various phytohormones, among which cytokinins (CK), which negatively regulate this process, play an important role. According to the results of our transcriptome analysis and real-time PCR of selected genes, the delayed leaf senescence in mutants with an inactivated gene of CK receptor AHK2 was associated with altered expression of trans-factor genes belonging to the families AP2-EREBP, bHLH, C2H2, GATA, MYB, NAC, and WRKY. The most significant quantitative changes in the ahk2 mutants, as compared to the leaves of wild type plants, were observed in the expression profile of the bHLH38, bHLH39, and bHLH100 genes from the pool of iron uptake regulators. Significantly increased levels of transcripts of these genes were associated with the activation of the FEP2 (AT1G47395) and FEP3 (AT1G47400) genes, capable of inducing the expression of bHLH38 and bHLH39 under conditions of iron deficiency. The increased expression of the DREB26, ESE3, GATA4, ETC2, and F11O4.13 genes was accompanied by an increased accumulation of transcripts of the AGP17 gene of arabinogalactan protein as well as the IBL1, BEE2, Ole e 1, and GDSL genes associated with the regulation of cell wall proteins. Thus, the AHK2 receptor, usually is atributed with an auxiliary role in the perception of the CK signal by leaves, is associated with a specific set of genes involved in the regulation of iron outflow and degradation of cell wall components at the final stages of leaf ontogenesis.

The inessential heavy metal/loids cadmium (Cd) and arsenic (As), which often co-occur in polluted paddy soils, are toxic to rice. Silicon (Si) treatment is known to reduce Cd and As toxicity in rice plants. To better understand the shared mechanisms by which Si alleviates Cd and As stress, rice seedlings were hydroponically exposed to Cd or As, then treated with Si. The addition of Si significantly ameliorated the inhibitory effects of Cd and As on rice seedling growth. Si supplementation decreased Cd and As translocation from roots to shoots, and significantly reduced Cd- and As-induced reactive oxygen species generation in rice seedlings. Transcriptomics analyses were conducted to elucidate molecular mechanisms underlying the Si-mediated response to Cd or As stress in rice. The expression patterns of the differentially expressed genes in Cd- or As-stressed rice roots with and without Si application were compared. The transcriptomes of the Cd- and As-stressed rice roots were similarly and profoundly reshaped by Si application, suggesting that Si may play a fundamental, active role in plant defense against heavy metal/loid stresses by modulating whole genome expression. We also identified two novel genes, Os01g0524500 and Os06g0514800, encoding a myeloblastosis (MYB) transcription factor and a thionin, respectively, which may be candidate targets for Si to alleviate Cd and As stress in rice, as well as for the generation of Cd- and/or As-resistant plants. This study provides valuable resources for further clarification of the shared molecular mechanisms underlying the Si-mediated alleviation of Cd and As toxicity in rice.

Synergid cells in the micropylar end of the female gametophyte are required for critical cell-cell signaling interactions between the pollen tube and the ovule that precede double fertilization and seed formation in flowering plants. LORELEI (LRE) encodes a GPI-anchored protein that is expressed primarily in the synergid cells, and together with FERONIA, a receptor-like kinase, it controls pollen tube reception by the receptive synergid cell. Still, how LRE expression is controlled in synergid cells remains poorly characterized. We identified candidate cis-regulatory elements enriched in LRE and other synergid cell-expressed genes. One of the candidate motifs (‘TAATATCT’) in the LRE promoter was an uncharacterized variant of the Evening Element motif that we named as the Short Evening Element-like (SEEL) motif. Deletion or point mutations in the SEEL motif of the LRE promoter resulted in decreased reporter expression in synergid cells, demonstrating that the SEEL motif is important for expression of LRE in synergid cells. Additionally, we found that LRE expression is decreased in the loss of function mutants of REVEILLE (RVE) transcription factors, which are clock genes known to bind the SEEL and other closely related motifs. We propose that RVE transcription factors regulate LRE expression in synergid cells by binding to the SEEL motif in the LRE promoter. Identification of a cis-regulatory element and transcription factors involved in the expression of LRE will serve as a foundation to characterize the gene regulatory networks in synergid cells and investigate the potential connection between circadian rhythm and fertilization.One sentence summaryA newly identified SEEL motif in the promoter of LORELEI and at least three members of the REVEILLE transcription factor family are important for LORELEI expression in synergid cells of the Arabidopsis female gametophyte.

Blackberry (Rubus spp.) fruit, contains high levels of anthocyanins in the pulp, is a good source of antioxidants. However, the molecular mechanisms underlying anthocyanin accumulation remains elusive. To improve our understanding of this process, we used RNA-seq to evaluate the transcriptional regulation of plant secondary metabolites, especially anthocyanin biosynthesis during fruit ripening. We constructed nine sequencing libraries, obtained 7.7 Gb clean bases and identified 50,563 annotated unigenes. Comparative transcriptome analysis revealed that 11,136, 33,268, and 31,913 genes were differentially expressed at Green vs. Red, Green vs. Black, and Red vs. Black, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment revealed that 35, 47, and 47 pathways were significantly enriched from these genes, respectively. We identified 58, 18, 24 and 48 differentially expressed genes (DEGs) involved in the anthocyanin biosynthesis, sucrose metabolism, abscisic acid (ABA), and ethylene (ETH) biosynthesis, respectively. Moreover, 66 DEGs of MYB were also identified in blackberry fruit. 24 DEGs from anthocyanin biosynthesis pathway and two DEGs of MYB were positively correlated with anthocyanin accumulation. Six DEGs of sucrose, six DEGs of ABA and seven DEGs of ETH were changed concomitantly with sucrose, ABA and ETH production. This study will provide an important information for understanding the molecular mechanism of anthocyanin accumulation during fruit ripening and a reference for related studies.

Maize, a monoecious crop, is a model plant for studying the regulation of floral organ development. In a previous study, we found that ZmMYB150 is significantly downregulated in sterile anthers by analyzing transcriptome data of the cytoplasmic male sterility (CMS) line C48-2 and its maintainer line 48-2. To elucidate the functional mechanism of ZmMYB150 in maize reproductive development, we cloned ZmMYB150, and sequence analysis showed it is a typical R2R3-MYB transcription factor (TF). Tissue-specific analysis revealed ZmMYB150 to be specifically highly in anthers. The subcellular localization in tobacco leaves proved that ZmMYB150 is located in the nucleus. Co-expression analysis identified 876 genes that may be coexpressed with ZmMYB150, and sequence alignment analysis showed that the coexpressed genes ZmMYB116 and ZmPIP5K6 are homologous to Arabidopsis male fertility-related genes. Furthermore, a yeast two-hybrid (Y2H) assay revealed that ZmMYB150 interacts with ZmPIP5K6. qRT-PCR analysis showed that ZmMYB150 is specifically highly expressed at the late stage of normal anther development, but no expression during the whole stages of sterile anther development. Tobacco transient expression system was used to confirm that ZmMYB150 can be regulated by miR159e-3p at the translational level. Our results lay a foundation for further understanding of the regulatory mechanism of MYB TFs in maize reproductive development.

Vitamin A deficiency is a worldwide public nutrition problem, and β-carotene is the precursor for vitamin A synthesis. Watermelon with golden flesh (gf, due to accumulated abundance of β-carotene) is an important germplasm resource. In this study, a genetic analysis of gf segregating populations indicated that gf was controlled by a single recessive gene. BSA-seq and an initial linkage analysis placed the gf locus in a 290-Kb region on watermelon chromosome 1. Further fine mapping in a large population with over 1,000 F2 plants narrowed this region to 39.08 Kb harboring two genes, Cla97C01G008760 and Cla97C01G008770, which encode phytoene synthase (ClPsy1) and GATA zinc finger domain-containing protein, respectively. Gene sequence alignment and expression analysis between parental lines revealed Cla97C01G008760 as the best possible candidate gene for gf trait. Nonsynonymous SNP mutations in the first exon of ClPsy1 between parental lines cosegregated with the gf trait only among individuals in the genetic population but were not related to flesh color in natural watermelon panels. Promoter sequence analysis of 26 watermelon accessions revealed two SNPs in the cis-acting element sequences corresponding to MYB and MYC2 transcription factors. RNA-seq data and qRT-PCR verification showed that two MYBs and one MYC2 exhibited expression trends similar as ClPsy1 in the parental lines, which may thus play roles in the regulation of ClPsy1 expression. Our research findings indicate that the gf trait is determined not only by ClPsy1 but also by ClLCYB, ClCRTISO and ClNCED7, which play important roles in β-carotene accumulation in watermelon flesh.

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.

Transcription factors (TFs) form the major class of regulatory genes and play key roles in multiple plant stress responses. In most eukaryotic plants, transcription factor (TF) families (WRKY, MADS-box and MYB) activate unique cellular-level abiotic and biotic stress-responsive strategies, which are considered as key determinants for defense and developmental processes. Arabidopsis and rice are two important representative model systems for dicot and monocot plants, respectively. A comprehensive comparative study on 101 OsWRKY, 34 OsMADS box and 122 OsMYB genes (rice genome) and, 71 AtWRKY, 66 AtMADS box and 144 AtMYB genes (Arabidopsis genome) showed various relationships among TFs across species. The phylogenetic analysis clustered WRKY, MADS-box and MYB TF family members into 10, 7 and 14 clades, respectively. All clades in WRKY and MYB TF families and almost half of the total number of clades in the MADS-box TF family are shared between both species. Chromosomal and gene structure analysis showed that the Arabidopsis-rice orthologous TF gene pairs were unevenly localized within their chromosomes whilst the distribution of exon–intron gene structure and motif conservation indicated plausible functional similarity in both species. The abiotic and biotic stress-responsive cis-regulatory element type and distribution patterns in the promoter regions of Arabidopsis and rice WRKY, MADS-box and MYB orthologous gene pairs provide better knowledge on their role as conserved regulators in both species. Co-expression network analysis showed the correlation between WRKY, MADs-box and MYB genes in each independent rice and Arabidopsis network indicating their role in stress responsiveness and developmental processes.

The ASYMMETRIC LEAVES2 (AS2) gene in Arabidopsis thaliana is responsible for the development of flat, symmetric, and extended leaf laminae and their vein systems. AS2 protein is a member of the plant-specific AS2/LOB protein family, which includes 42 members comprising the conserved amino-terminal domain referred to as the AS2/LOB domain, and the variable carboxyl-terminal region. Among the members, AS2 has been most intensively investigated on both genetic and molecular levels. AS2 forms a complex with the myb protein AS1, and is involved in epigenetic repression of the abaxial genes ETTIN/AUXIN RESPONSE FACTOR3 (ETT/ARF3), ARF4, and class 1 KNOX homeobox genes. The repressed expression of these genes by AS2 is markedly enhanced by the cooperative action of various modifier genes, some of which encode nucleolar proteins. Further downstream, progression of the cell division cycle in the developing organs is stimulated; meristematic states are suppressed in determinate leaf primordia; and the extension of leaf primordia is induced. AS2 binds the specific sequence in exon 1 of ETT/ARF3 and maintains methylated CpGs in several exons of ETT/ARF3. AS2 forms bodies (designated as AS2 bodies) at nucleolar peripheries. AS2 bodies partially overlap chromocenters, including inactive 45S ribosomal DNA repeats, suggesting the presence of molecular and functional links among AS2, the 45S rDNAs, and the nucleolus to exert the repressive regulation of ETT/ARF3. The AS2/LOB domain is characterized by three subdomains, the zinc finger (ZF) motif, the internally conserved-glycine containing (ICG) region, and the leucine-zipper-like (LZL) region. Each of these subdomains is essential for the formation of AS2 bodies. ICG to LZL are required for nuclear localization, but ZF is not. LZL intrinsically has the potential to be exported to the cytoplasm. In addition to its nuclear function, it has been reported that AS2 plays a positive role in geminivirus infection: its protein BV1 stimulates the expression of AS2 and recruits AS2 to the cytoplasm, which enhances virus infectivity by suppression of cytoplasmic post transcriptional gene silencing.

Wheat powdery mildew (Pm) caused by Blumeria graminis f. sp. tritici (Bgt) is a widespread epidemic disease in wheat. Haynaldia villosa, a diploid wild relative of wheat, has been widely utilized in wheat breeding for Pm resistance as a broad-spectrum resistance (BSR) resource. A previous study reported a Serine/threonine protein kinase-V (Stpk-V) gene from H. villosa showing BSR to wheat Pm. To deduce the defense network regulated by Stpk-V, we performed temporal RNA-seq of Stpk-V transgenic line (OEStpk-V) and its receptor wheat variety Yangmai 158 during Bgt infection. Gene Ontology enrichment indicated ROS were rapidly activated in OEStpk-V at 1 h after inoculation (hai). The qRT-PCR confirmed the upregulated transcription of ROS related genes OxO and POD, and suppression of ROS scavenging genes SOD and CAT. Besides, four transcription factors genes (WRKY, MYB, ERF, NAC) related to the ROS defense pathway were also enriched. Kyoto encyclopedia of genes and genomes enrichment revealed a remarkable reprogramming of the Ca2+ signalling pathway in OEStpk-V at 1 and 3 hai. qRT-PCR confirmed significantly induced transcription of CDPKs, CALMs, CMLs, and CNGCSs in OEStpk-V. Weighted gene co-expression network analysis classified Stpk-V to the same module in which the Ca2+ signalling related genes were included. Our results indicated that the reprogramming of the ROS accumulation and Ca2+ signalling at initial infection stages plays a crucial role in the Stpk-V regulatory network during wheat-Bgt interaction.

Petals of the monocot Phalaenopsis aphrodite (Orchidaceae) possess conical epidermal cells on their adaxial surfaces, and a large amount of cuticular wax is deposited on them to serve as a primary barrier against biotic and abiotic stresses. It has been widely reported that subgroup 9A members of the R2R3-MYB gene family, MIXTA and MIXTA-like in eudicots, act to regulate the differentiation of conical epidermal cells. However, the molecular pathways underlying conical epidermal cell development and cuticular wax biosynthesis in monocot petals remain unclear. Here, we characterized two subgroup 9A R2R3-MYB genes, PaMYB9A1 and PaMYB9A2 (PaMYB9A1/2), from P. aphrodite through the transient overexpression of their coding sequences and corresponding chimeric repressors in developing petals. We showed that PaMYB9A1/2 function to coordinate conical epidermal cell development and cuticular wax biosynthesis. In addition, we identified putative targets of PaMYB9A1/2 through comparative transcriptome analyses, revealing that PaMYB9A1/2 acts to regulate the expression of cell wall-associated and wax biosynthetic genes. Furthermore, a chemical composition analysis of cuticular wax showed that even-chain n-alkanes and odd-chain primary alcohols are the main chemical constituents of cuticular wax deposited on petals, which is inconsistent with the well-known biosynthetic pathways of cuticular wax, implying a distinct biosynthetic pathway occurring in P. aphrodite flowers. These results reveal that the function of subgroup 9A R2R3-MYB family genes in regulating the differentiation of epidermal cells is largely conserved in monocots and dicots. Furthermore, both PaMYB9A1/2 have evolved additional functions controlling the biosynthesis of cuticular wax., Two subgroup 9A R2R3-MYB genes act to regulate the development of conical epidermal cells and the biosynthesis of cuticular wax in Phalaenopsis petals.

Two MYB genes have been identified which regulate apocarotenoid metabolism in Crocus sativus. Apocarotenoids like crocin, picrocrocin and safranal are restricted to genus Crocus and are synthesized by oxidative cleavage of zeaxanthin followed by glycosylation reactions. In Crocus sativus, these apocarotenoids are synthesized in stigma part of the flower in developmentally regulated manner. Most of the genes of apocarotenoid pathway are known, however, the mechanism that regulates its tissue and stage specific biosynthesis remains elusive. MYB family was identified as the largest transcription factor family from Crocus transciptome which indicated its possible role in apocarotenoid regulation besides regulating other metabolic pathways. Towards this, we started with identification of 150 MYB genes from Crocus transcriptome databases. The phylogenetic analysis of Crocus MYB genes divided them into 27 clusters. Domain analysis resulted in identification of four groups of MYBs depending upon the number of R repeats present. Expression profiling indicated that 12 MYBs are upregulated in stigma out of which expression of four genes CstMYB1, CstMYB14, CstMYB16 and CstMYB1R2 correlated with crocin accumulation. Transient overexpression of two nuclear localized MYB genes (CstMYB1 and CstMYB1R2) in Crocus confirmed their role in regulating carotenoid metabolism. Yeast-one-hybrid confirmed that CstMYB1 binds to carotenoid cleavage dioxygenase 2 (CCD2) promoter while CstMYB1R2 binds to phytoene synthase (PSY) and CCD2 promoters. Overall, our study established that CstMYB1 and CstMYB1R2 regulate apocarotenoid biosynthesis by directly binding to promoters of pathway genes.

Cotton is an important fibre-producing crop. Cotton fibres consist of highly elongated trichomes derived from the ovule. To improve the quality of cotton, it is necessary to identify the genes regulating fibre development. GhMYB3 was identified through bioinfomatic analysis and introduced to Arabidopsis and cotton to observe the phenotype. Protein inteaction and promoter bingding assays were conducted to explore the role of GhMYB3 in trichome fibre growth. Cotton fibre development might share a similar regulatory mechanism to Arabidopsis leaf trichomes, which is determined by the essential regulatory complex, MYB-bHLH-WD40. The GL1-like R2R3 MYB transcription factor GhMYB3 interacts with the AtGL3 protein involved in Arabidopsis trichome development. Ectopic expression of GhMYB3 could rescue the glabrous phenotype of the Arabidopsis gl1 mutant and produced more ectopic trichomes on inflorescence stems and floral organs, confirming its orthologous function in plant trichome development. The expression of GhMYB3 increased in response to exogenous gibberellin (GA3 ), auxin (IAA) and methyl jasmonate (MeJA). Overexpression of this gene in cotton leads to a slight increase in fibre length and lint percentage, possibly by activating the transcription of its downstream gene GhRDL1 or other fibre-related genes. The results increase our understanding of the key role of GhMYB3 in positively controlling plant trichome development, and this gene could be a potential target for molecular breeding in cotton.

Seed germination is critical for plant survival and agricultural production, which is affected by both internal seed factors and external environmental conditions. However, the genetic basis and underlying molecular mechanisms of early seed germination in crops remain largely unclear. Here, we report that R2R3 MYB transcription factor Carbon Starved Anther (CSA) is expressed specifically in Oryza sativa embryo and aleurone in response to seed imbibition, peaking at 3–6 h and undetectable by 24-h post-imbibition. CSA seeds germinated more quickly than wild-type rice seeds and had higher levels of amylase activity, glucose, and inactive abscisic acid-glucose ester (ABA-GE), but lower levels of ABA. Through analyzing the CSA-associated transcriptome and CSA binding to downstream target genes, we identified two glycolytic genes as direct CSA targets. CSA inhibits Amylase 3A expression to limit glucose production from starch and activates Os3BGlu6 expression to promote de-conjugation of ABA-GE to ABA; these functions serve to slow germination and improve seedling resilience to abiotic stress in the first 3 weeks of growth. Therefore, this study unveils a protection mechanism conferred by CSA during early seed germination by balancing glucose and ABA metabolism to optimize seed germination and stress response fitness.

Spinach (Spinacia oleracea L.) is commonly considered a dioecious plant with heterogametic (XY) and homogametic (XX) sex chromosomes. The characteristic is also utilized for the production of spinach hybrid seeds. However, the molecular mechanisms of sex determination in spinach are still unclear because of a lack of genomic and transcriptomic information. In this study, RNA sequencing (RNA-seq) was performed in male and female inflorescences to provide insight into the molecular basis of sex determination in spinach. Comparative transcriptome analyses showed that 2278 differentially expressed genes (DEGs) were identified between male and female inflorescences. A high correlation between the RNA-Seq and qRT-PCR validation for DEGs was observed. Among these, 182 DEGs were annotated to transcription factors including the MYB family protein, bHLH family, and MADS family, suggesting these factors might play a vital role in sex determination. Moreover, 26 DEGs related to flower development, including nine ABCE class genes, were detected. Expression analyses of hormone pathways showed that brassinosteroids may be key hormones related to sex determination in spinach. Overall, this study provides a large amount of DEGs related to sexual expression and lays a foundation for unraveling the regulatory mechanism of sex determination in spinach.

Mechanisms associated with the control of flower color in crape myrtle varieties have yet to be sufficiently elucidated, which has tended to hamper the use of modern molecular and genetic strategies in the breeding programs for this plant. The whole transcriptome of four L. indica varieties characterized by different flower colors (white, light purple, deep purplish pink, and strong red) was sequenced, and we performed bioinformatic, quantitative PCR, and co-expression analyses of R2R3 MYB transcription factor and anthocyanin/flavonol pathway genes. We obtained a total of 49,980 transcripts with full-length coding sequences. Both transcriptome and qPCR analyses revealed that anthocyanin/flavonol pathway genes were differentially expressed among the four different flowers types, with the expression of LiPAL, LiCHS, LiCHI, LiDFR, LiANS/LDOX, and LiUFGT being induced in colorful flowers, whereas that of LiF3´5´H, LiFLS, and LiLAR was found to be inhibited. Base on phylogenetic analysis, seven R2R3 MYB transcriptional factors were identified as putative regulators of flower color. The molecular characteristics and co-expression patterns indicated that these MYBs differentially modulate their target genes, with two probably acting as activators, three as repressors, and one contributing to the regulation of vacuolar pH. The findings of this study indicate that the anthocyanin biosynthesis is more active than the flavonol and proanthocyanin in the colorful flowers. These observations provide new genomic information on L. indica and contribute gene resources for the flower color-targeted breeding of crape myrtle.

The MYB transcription factors play versatile roles in regulating secondary metabolites in plants, especially, in phenylpropane metabolism and anthocyanin biosynthesis. However, research on MYB transcription factors regulation of other metabolic pathways is rare. Here, we report the effects of constitutive expression of the MYB transcription factor LrAN2 from Lycium ruthenicum in potato. Overexpression of LrAN2 in potato led to an increase in the accumulation of anthocyanins and simultaneously altered the profile of the total glycoalkaloids (SGAs). Overexpression of LrAN2 upregulated the expression of related genes in the anthocyanin biosynthetic pathway and altered the expression of related genes in the SGA biosynthetic pathway in potato. Our findings demonstrated that LrAN2 might engage in crosstalk between the accumulation of anthocyanins and SGAs in potato leaves, offering a new breeding strategy for potato improvements. The MYB transcription factor LrAN2 from Lycium ruthenicum regulates the biosynthesis of anthocyanins and glycoalkaloids in potato.

Key message Identification and functional analysis of the male sterile gene MS6 in Glycine max. Abstract Soybean (Glycine max (L.) Merr.) is an important crop providing vegetable oil and protein. The male sterility-based hybrid breeding is a promising method for improving soybean yield to meet the globally growing demand. In this research, we identified a soybean genic male sterile locus, MS6, by combining the bulked segregant analysis sequencing method and the map-based cloning technology. MS6, highly expressed in anther, encodes an R2R3 MYB transcription factor (GmTDF1-1) that is homologous to Tapetal Development and Function 1, a key factor for anther development in Arabidopsis and rice. In male sterile ms6 (Ames1), the mutant allele contains a missense mutation, leading to the 76th leucine substituted by histidine in the DNA binding domain of GmTDF1-1. The expression of soybean MS6 under the control of the AtTDF1 promoter could rescue the male sterility of attdf1 but ms6 could not. Additionally, ms6 overexpression in wild-type Arabidopsis did not affect anther development. These results evidence that GmTDF1-1 is a functional TDF1 homolog and L76H disrupts its function. Notably, GmTDF1-1 shows 92% sequence identity with another soybean protein termed as GmTDF1-2, whose active expression also restored the fertility of attdf1. However, GmTDF1-2 is constitutively expressed at a very low level in soybean, and therefore, not able to compensate for the MS6 deficiency. Analysis of the TDF1-involved anther development regulatory pathway showed that expressions of the genes downstream of TDF1 are significantly suppressed in ms6, unveiling that GmTDF1-1 is a core transcription factor regulating soybean anther development.

Lonicera japonica Thunb., a traditional Chinese herb, has been used for treating human diseases for thousands of years. Recently, the genome of L. japonica has been decoded, providing valuable information for research into gene function. However, no comprehensive database for gene functional analysis and mining is available for L. japonica. We therefore constructed LjaFGD (www.gzybioinformatics.cn/LjaFGD and bioinformatics.cau.edu.cn/LjaFGD), a database for analyzing and comparing gene function in L. japonica. We constructed a gene co-expression network based on 77 RNA-seq samples, and then annotated genes of L. japonica by alignment against protein sequences from public databases. We also introduced several tools for gene functional analysis, including Blast, motif analysis, gene set enrichment analysis, heatmap analysis, and JBrowse. Our co-expression network revealed that MYB and WRKY transcription factor family genes were co-expressed with genes encoding key enzymes in the biosynthesis of chlorogenic acid and luteolin in L. japonica. We used flavonol synthase 1 (LjFLS1) as an example to show the reliability and applicability of our database. LjaFGD and its various associated tools will provide researchers with an accessible platform for retrieving functional information on L. japonica genes to further biological discovery.

As one of the most popular bulbous plant with high ornamental value, Lilium spp. has been used as cut flower or green plants in urban landscapes and home gardening. We noticed a cultivar ‘Elodie’ show weak double-flowered phenotype, by which whole 3 can be classified into three different types including stamen, partially petaloid stamen (PPetSta) and totally petaloid stamen (TPetSta). To understand the molecular basis of this homeotically conversion variation, quantitative RNA-Seq analysis was conducted to screen the important unigenes underlying this phenomenon. Meanwhile, endogenous hormone contents including auxin, cytokinin, GA and ABA were detected at the same time. In total, we assembled and annotated 53,182 unigenes. 8453, 21,281 and 7646 DEGs (differentially expressed genes) were identified from PPetSta vs Stamen, TPetSta vs Stamen, and TPetSta vs PPetSta comparisons, in which 2241 unigenes were shared DEGs. 20 transcription factors in MADS-box and MYB families, 43 unigenes in signaling and synthesis pathways of auxin, cytokinin, GA, ABA and ethylene were differentially expressed. Meanwhile, endogenous hormone contents including auxin, cytokinin, GA and ABA were declined in partially petaloidy flower and totally petaloidy flower when compared with the normal stamen. The expression of GA20ox3 and CYP735A2 encode the biosynthesis genes of GA and cytokinin, respectively were downregulated, which was consistent with the hormone determination results. The expression of SAUR50, SLR5, PYL9, ERF4/27 involved in the signaling pathways of IAA, GA, ABA, ethylene and transcription factors MYB44/108, AG2, AGL104, MGS1 had the same expression profiles corresponding with the endogenous hormone content, indicating that they may play a positive role during the process of stamen petalization. This work will help us to unravel several specific genes and provide a theoretical basis for double flower breeding of Lilium species.

Background Flavonoid biosynthesis in plants is primarily regulated at the transcriptional level by transcription factors modulating the expression of genes encoding enzymes in the flavonoid pathway. One of the most studied transcription factor complexes involved in this regulation consists of a MYB, bHLH and WD40. However, in Chinese Narcissus (Narcissus tazetta L. var. chinensis), a popular monocot bulb flower, the regulatory mechanism of flavonoid biosynthesis remains unclear. Results In this work, genes related to the regulatory complex, NtbHLH1 and a R2R3-MYB NtMYB6, were cloned from Chinese Narcissus. Phylogenetic analysis indicated that NtbHLH1 belongs to the JAF13 clade of bHLH IIIf subgroup, while NtMYB6 was highly homologous to positive regulators of proanthocyanidin biosynthesis. Both NtbHLH1 and NtMYB6 have highest expression levels in basal plates of Narcissus, where there is an accumulation of proanthocyanidin. Ectopic over expression of NtbHLH1 in tobacco resulted in an increase in anthocyanin accumulation in flowers, and an up-regulation of expression of the endogenous tobacco bHLH AN1 and flavonoid biosynthesis genes. In contrast, the expression level of LAR gene was significantly increased in NtMYB6-transgenic tobacco. Dual luciferase assays showed that co-infiltration of NtbHLH1 and NtMYB6 significantly activated the promoter of Chinese Narcissus DFR gene. Furthermore, a yeast two-hybrid assay confirmed that NtbHLH1 interacts with NtMYB6. Conclusions Our results suggest that NtbHLH1 may function as a regulatory partner by interacting directly with NtMYB6 to enhance proanthocyanidin accumulation in Chinese Narcissus.

Telomeric repeat binding factor a (Terfa) derived from zebrafish is a homologous protein with human telomeric repeat binding factor 2 (TRF2). Terfa is known as a senescence-associated biomarker in various research through the zebrafish animal model. In addition, according to the findings so far, it has been confirmed that human or plant telomere binding proteins bind to telomeric DNA specialized for each species, but, in our result, Terfa shows it strongly binds to both human or plant type telomeric DNA. Here we characterized the DNA binding properties and demonstrate the solution structure of Terfa and identified residues participating in the interaction with both human and plant telomeric DNA. In DNA recognition of human and plant telomere binding proteins, the N-terminal loop and the α-helix 3 part of Myb domain were bound majorly, whereas, in the case of Terfa, the N-terminal loop, the α-helix 1-2 loop, and α-helix 2 of the Myb domain were dominantly bound. Therefore, when Terfa recognizes DNA, it was found that the binding module differs from previously known telomere binding proteins. The comparison of the structure of the telomere binding proteins provides an opportunity to understand more specifically how the structural properties of each telomere binding protein are associated with telomeric DNA binding from an evolutionary point of view.

UV-B radiation is a pivotal photomorphogenic signal and positively regulates plant growth and metabolite biosynthesis. In order to elucidate the transcriptional regulation mechanism underlying UV-B-induced artemisinin and flavonoid biosynthesis in Artemisia annua, the transcriptional responses of A. annua L. leaves to UV-B radiation were analyzed using the Illumina transcriptome sequencing. A total of 10705 differentially expressed genes (DEGs) including 533 transcription factors (TFs), were identified. Based on the expression trends of the differentially expressed TFs as well as artemisinin and flavonoid biosynthesis genes, we speculated that TFs belonging to 6 clusters were most likely to be involved in the regulation of artemisinin and/or flavonoid biosynthesis. The regulatory relationship between TFs and artemisinin/flavonoid biosynthetic genes was further studied. Dual-LUC assays results showed that AaMYB6 is a positive regulator of AaLDOX which belongs to flavonoid biosynthesis pathway. In addition, we identified an R2R3 MYB TF, AaMYB4 which potentially mediated both artemisinin and flavonoid biosynthesis pathways by activating the expression of AaADS and AaDBR2 in artemisinin biosynthesis pathway and AaUFGT in flavonoid biosynthesis pathway. Overall, our findings would provide an insight into the elucidation of the parallel transcriptional regulation of artemisinin and flavonoid biosynthesis in A. annua L. under UV-B radiation.

Flower development exists as a key period in the angiosperms life cycle and the proper development is considered with its reproductive success. Pistil abortion is one of the widely distributed aspects of berry plants and its basic mechanism in Japanese apricot is quite unclear and needs thorough investigation. The present study was carried out to get a deep insight into the pistil abortion mechanism in Japanese apricot using a transcriptomic approach. A large number of DEGs were identified from different development stages of normal and abortive pistils. Pair-wise comparison analysis was performed as LY1 vs DQD1, LY2 vs DQD2, and LY3 vs DQD3 and produced 3590, 2085, and 2286 transcripts, respectively. The Gene Ontology (GO) showed that different metabolic processes, plant hormones, developmental processes, and photosystem-related genes were involved in pistil abortion. The pathway analysis revealed significant enrichment of plant hormone's signal transduction and circadian rhythm pathways. Furthermore, transcription factors such as MYB, MADS-box, and NAC family showed lower expression in abortive pistils. The current study presents a new strategy for advanced research and understanding of the pistil abortion process in Japanese apricot and provides a possible reference for other deciduous fruit trees.

Background Polyploid plants often exhibit enhanced stress tolerance. The underlying physiological and molecular bases of such mechanisms remain elusive. Here, we characterized the drought tolerance of autotetraploid sour jujube at phenotypic, physiological and molecular levels. Results The study findings showed that the autotetraploid sour jujube exhibited a superior drought tolerance and enhanced regrowth potential after dehydration in comparison with the diploid counterpart. Under drought stress, more differentially expressed genes (DEGs) were detected in autotetraploid sour jujube and the physiological responses gradually triggered important functions. Through GO enrichment analysis, many DEGs between the diploid and autotetraploid sour jujube after drought-stress exposure were annotated to the oxidation–reduction process, photosystem, DNA binding transcription factor activity and oxidoreductase activity. Six reactive oxygen species scavenging-related genes were specifically differentially expressed and the larger positive fold-changes of the DEGs involved in glutathione metabolism were detected in autotetraploid. Consistently, the lower O2− level and malonaldehyde (MDA) content and higher antioxidant enzymes activity were detected in the autotetraploid under drought-stress conditions. In addition, DEGs in the autotetraploid after stress exposure were significantly enriched in anthocyanin biosynthesis, DNA replication, photosynthesis and plant hormone, including auxin, abscisic acid and gibberellin signal-transduction pathways. Under osmotic stress conditions, genes associated with the synthesis and transport of osmotic regulators including anthocyanin biosynthesis genes were differentially expressed, and the soluble sugar, soluble protein and proline contents were significantly higher in the autotetraploid. The higher chlorophyll content and DEGs enriched in photosynthesis suggest that the photosynthetic system in the autotetraploid was enhanced compared with diploid during drought stress. Moreover, several genes encoding transcription factors (TFs) including GRAS, Bhlh, MYB, WRKY and NAC were induced specifically or to higher levels in the autotetraploid under drought-stress conditions, and hub genes, LOC107403632, LOC107422279, LOC107434947, LOC107412673 and LOC107432609, related to 18 up-regulated transcription factors in the autotetraploid compared with the diploid were identified. Conclusion Taken together, multiple responses contribute to the enhanced drought tolerance of autotetraploid sour jujube. This study could provide an important basis for elucidating the mechanism of tolerance variation after the polyploidization of trees.

Walnut is a popular nut tree species and usually suffers from drought stress. However, little information is available on the mechanism of walnut responding to drought stress, resulting in lack of basic understanding for its resistance. In order to excavate more functional genes that can respond to stressors, and enrich the theoretical basis for walnut resistance, in this study, 5 MYB genes with complete ORFs were identified from J. regia and the basic bio-information as well as expression patterns in different tissues and response to drought and ABA stresses were confirmed using qRT-PCR assay. The results showed that 2 JrMYB genes belong to R1-MYB subfamily and 3 JrMYBs belong to R2R3-MYB, encoding the proteins from 212 to 362 aa in length. The phylogenetic analysis categorized proteins of 5 JrMYBs and 40 Arabidopsis AtMYBs into 10 subgroups. JrMYBs in the same subgroup exhibited significant similarities in the composition of conserved domains and motifs in amino acid sequences and exon/intron organization in DNA sequences. The results of qRT-PCR analysis revealed that JrMYB genes diversely expressed in various tissues. Moreover, the expression values of JrMYBs were upregulated or downregulated significantly under drought and ABA stresses. Most attractively, in contrast with suffering from drought stress alone, the treatments with drought and additional ABA greatly enhanced the transcript levels of JrMYBs. All these results suggested that JrMYB genes play a vital role in plant biological processes and drought as well as ABA stress response, and possibly perform as ABA-dependent drought response transcription factors in plant.

AimsDrought is a climate threat limiting crop production. Potato is one of the four most important food crops worldwide and is sensitive to water shortage. The CBP80 gene was shown to affect plant response to drought by regulating the level of microRNA159, and, consequently, the levels of the MYB33 and MYB101 transcription factors. (TF) Our studies aimed to show whether indeed the level of MYB33, MYB65, and MYB101 TFs affects plant response to water shortage.MethodsArabidopsis transgenic plants exhibiting downregulation and Arabidopsis and potato transgenic plants exhibiting overexpression of selected MYB TFs were obtained. Plants response to drought was mainly measured using relative water content (RWC) and stomata closure upon exogenous ABA.ResultsThree MYB TFs studied are involved in plant response to drought. When downregulated in Arabidopsis, the MYB33, MYB65 and MYB101 genes cause stomatal hyposensitivity to ABA, leading to reduced tolerance to drought. Transgenic Arabidopsis and potato plants overexpressing a mutated version of these genes with changed miR159 recognition site, show hypersensitivity to ABA and relatively high tolerance to drought conditions.ConclusionsThe MYB33, MYB65, and MYB101 genes are good be potential targets for innovative breeding to obtain crops with relatively high tolerance to drought.

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.

Telomere repeat-containing RNA (TERRA) has been identified in multiple organisms including Trypanosoma brucei, a protozoan parasite that causes human African trypanosomiasis. T. brucei regularly switches its major surface antigen, VSG, to evade the host immune response. VSG is expressed exclusively from subtelomeric expression sites, and we have shown that telomere proteins play important roles in the regulation of VSG silencing and switching. In this study, we identify several unique features of TERRA and telomere biology in T. brucei. First, the number of TERRA foci is cell cycle-regulated and influenced by TbTRF, the duplex telomere DNA binding factor in T. brucei. Second, TERRA is transcribed by RNA polymerase I mainly from a single telomere downstream of the active VSG. Third, TbTRF binds TERRA through its C-terminal Myb domain, which also has the duplex DNA binding activity, in a sequence-specific manner and suppresses the TERRA level without affecting its half-life. Finally, levels of the telomeric R-loop and telomere DNA damage were increased upon TbTRF depletion. Overexpression of an ectopic allele of RNase H1 that resolves the R-loop structure in TbTRF RNAi cells can partially suppress these phenotypes, revealing an underlying mechanism of how TbTRF helps maintain telomere integrity.

Phytosulfokine (PSK) receptor signaling promotes root elongation, determines lateral root density, and maintains non-hair cell fate partially independent of TPST responsible for the activating sulfation of PSK., Tyrosine-sulfated peptides are key regulators of plant growth and development. The disulfated pentapeptide phytosulfokine (PSK) mediates growth via leucine-rich repeat receptor-like kinases, PSKR1 and PSKR2. PSK receptors (PSKRs) are part of a response module at the plasma membrane that mediates short-term growth responses, but downstream signaling of transcriptional regulation remains unexplored. In Arabidopsis, tyrosine sulfation is catalyzed by a single-copy gene (TPST; encoding tyrosylprotein sulfotransferase). We performed a microarray-based transcriptome analysis in the tpst-1 mutant background that lacks sulfated peptides to identify PSK-regulated genes and genes that are regulated by other sulfated peptides. Of the 169 PSK-regulated genes, several had functions in root growth and development, in agreement with shorter roots and a higher lateral root density in tpst-1. Further, tpst-1 roots developed higher numbers of root hairs, and PSK induced expression of WEREWOLF (WER), its paralog MYB DOMAIN PROTEIN 23 (MYB23), and At1g66800 that maintain non-hair cell fate. The tpst-1 pskr1-3 pskr2-1 mutant showed even shorter roots, and higher lateral root and root hair density than tpst-1, revealing unexpected synergistic effects of ligand and PSKR deficiencies. While residual activities may exist, overexpression of PSKR1 in the tpst-1 background induced root growth, suggesting that PSKR1 may be active in the absence of sulfated ligands.

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.

The bacterial leaf blight in rice caused by Xanthomonas oryzae pv. oryzae (Xoo) affects crop losses worldwide. In spite of developing resistant varieties by introgressing different Xa genes, the occurrence of diseases is evident. Here we report identification of several genes that are associated with improved plant immunity against Xoo in a resistant genotype BPT-5204 in comparison with susceptible genotype TN-1. The RNA sequencing information was developed to identify the genes that could provide durable resistance in rice. Xoo-resistant rice genotype BPT-5204 with Xa 5, 13 and 21 genes is compared with sensitive Taichung Native 1 (TN-1) to identify the genetic pathways and gene networks involved in resistance mechanisms. The higher levels of salicylic acid resulted in upregulation of many pathogenesis-related (PR) and redox protein encoding transcripts which resulted in higher hypersensitive response in BPT-5204. Many Serine/threonine protein kinase, leucine-rich repeat (LRR) transmembrane protein kinase, protein kinase family genes, Wall-associated kinase (WAK) were upregulated that resulted in activation of bZIP, WRKY, MYB, DOF and HSFs transcription factors that are associated with improved plant immunity. The study provided roles of many genes and their associated plant immunity pathways that can be used for developing resistant rice cultivars.

Sophora davidii is an ideal material for studying the response to low phosphorus (P) because of its strong adaptability to low-P habitats. However, to date, there has been no research on S. davidii under low-P stress. In this paper, the differentially expressed genes (DEGs) of S. davidii were studied on days 3 and 9 of low-P treatment by transcriptome sequencing and analysed with GO and KEGG analyses. Under low-P stress, the contents of antioxidants (POD, SOD and CAT), osmotic adjusters (soluble protein and proline) and anthocyanin increased. Under low-P and normal-P conditions, 323 (209 and 114 of whose expression was upregulated and downregulated, respectively) and 504 DEGs (280 and 224 of whose expression was upregulated and downregulated, respectively) were found in S. davidii on days 3 and 9, respectively. The analysis of transcriptomics data revealed certain aspects of genes regulation under low-P stress. First, several key candidate genes, such as Orphans, GNAT, bZIP, MYB, AP2-EREBP, NAC and WRKY genes, were predicted to determine tolerance to low-P stress. Second, four pathways (starch and sucrose metabolism; plant hormone signal transduction; phenylpropanoid biosynthesis; and alanine, aspartate and glutamate metabolism) were correlated with low-P stress and were highly differentially regulated in two time treatments. These results provide new insights into the complex mechanisms of the low-P response in S. davidii or even other plant species.

Commonly found flavonols in plants are synthesized from dihydroflavonols by flavonol synthase (FLS). The genome of Arabidopsis thaliana contains six FLS genes, among which FLS1 encodes a functional enzyme. Previous work has demonstrated that the R2R3-MYB subgroup 7 transcription factors MYB11, MYB12, and MYB111 redundantly regulate flavonol biosynthesis. However, flavonol accumulation in pollen grains was unaffected in the myb11myb12myb111 triple mutant. Here we show that MYB21 and its homologs MYB24 and MYB57, which belong to subgroup 19, promote flavonol biosynthesis through regulation of FLS1 gene expression. We used a combination of genetic and metabolite analysis to identify the role of MYB21 in regulating flavonol biosynthesis through direct binding to the GARE cis-element in the FLS1 promoter. Treatment with kaempferol or overexpression of FLS1 rescued stamen defects in the myb21 mutant. We also observed that excess reactive oxygen species (ROS) accumulated in the myb21 stamen, and that treatment with the ROS inhibitor diphenyleneiodonium chloride partly rescued the reduced fertility of the myb21 mutant. Furthermore, drought increased ROS abundance and impaired fertility in myb21, myb21myb24myb57, and chs, but not in the wild type or myb11myb12myb111, suggesting that pollen-specific flavonol accumulation contributes to drought-induced male fertility by ROS scavenging in Arabidopsis., MYB21 modulates the accumulation of flavonols through directly binding to the GARE cis-element of the FLS1 promoter, which is essential for the reduced-fertility phenotype of the myb21 mutant.

Background Melatonin, a multifunctional signal molecule, has been reported to play crucial roles in growth and development and stress responses in various plant species. Okra (Abelmoschus esculentus L.) is a food crop with extremely high values of nutrition and healthcare. Recent reports have revealed the protective role of melatonin in alleviating salt stress. However, little is known about its regulatory mechanisms in response to salt stress in okra. Results In this study, we explored whether exogenous melatonin pretreatment could alleviate salt stress (300 mM NaCl) of okra plants. Results showed that exogenous application of melatonin (50 μM) significantly enhanced plant tolerance to salt stress, as demonstrated by the plant resistant phenotype, as well as by the higher levels of the net photosynthetic rate, chlorophyll fluorescence and chlorophyll content in comparison with nontreated salt-stressed plants. Additionally, melatonin pretreatment remarkably decreased the levels of lipid peroxidation and H2O2 content and scavenged O2•- in melatonin-pretreated plants, which may be attributed to the higher levels of enzyme activities including POD and GR. Moreover, a combination of third- (PacBio) and second-generation (Illumina) sequencing technologies was applied to sequence full-length transcriptomes of okra. A total of 121,360 unigenes was obtained, and the size of transcript lengths ranged from 500 to 6000 bp. Illumina RNA-seq analysis showed that: Comparing with control, 1776, 1063 and 1074 differential expression genes (DEGs) were identified from the three treatments (NaCl, MT50 and MT + NaCl, respectively). These genes were enriched in more than 10 GO terms and 34 KEGG pathways. Nitrogen metabolism, sulfur metabolism, and alanine, aspartate and glutamate metabolism were significantly enriched in all three treatments. Many transcription factors including MYB, WRKY, NAC etc., were also identified as DEGs. Conclusions Our preliminary results suggested that melatonin pretreatment enhanced salt tolerance of okra plants for the first time. These data provide the first set of full-length isoforms in okra and more comprehensive insights into the molecular mechanism of melatonin responses to salt stress.

Key enzymes play a vital role in plant growth and development. However, the evolutionary relationships between genes encoding key enzymes in the metabolic pathway of Tartary buckwheat flavonoids are poorly understood. Based on the published Tartary buckwheat genome sequence and related Tartary buckwheat transcriptome data, 48 key enzyme-encoding genes involved in flavonoid metabolism were screened from the Tartary buckwheat genome in this study; the chromosome localization, gene structure and promoter elements of these enzyme-encoding gene were also investigated. Gene structure analysis revealed relatively conserved 5' exon sequences among the 48 genes, indicating that the structural diversity of key enzyme-encoding genes is low in Tartary buckwheat. Through promoter analysis, these key enzyme-encoding genes were found to contain a large number of light-response elements and hormone-response elements. In addition, some genes could bind MYB transcription factors, participating in the regulation of flavonoid biosynthesis. The transcription level of the 48 key enzyme-encoding gene varied greatly among tissues. In this study, we identified 48 key enzyme-encoding genes involved in flavonoid metabolic pathways, and elucidated the structure, evolution and tissue-specific expression patterns of these genes. These results lay a foundation for further understanding the functional characteristics and evolutionary relationships of key enzyme-encoding genes involved in the flavonoid metabolic pathway in Tartary buckwheat.

The anthocyanin synthetic pathway is regulated centrally by an MYB-bHLH-WD40 (MBW) complex. Anthocyanin pigmentation is an important fruit quality trait in red-fleshed kiwifruit; however, the underlying regulatory mechanisms involving the MBW complex are not well understood. In this study, one R2R3MYB (AcMYBF110 expressed in fruit characteristically), one bHLH (AcbHLH1), two upstream regulators of AcbHLH1 (AcbHLH4 and AcbHLH5), and one WDR (AcWDR1) are characterized as being involved in the regulation of anthocyanin synthesis in kiwifruit. AcMYBF110 plays an important role in the regulation of anthocyanin accumulation by specifically activating the promoters of several anthocyanin pathway genes including AcCHS, AcF3'H, AcANS, AcUFGT3a, AcUFGT6b, and AcGST1. Coexpression of AcbHLH1, AcbHLH4, or AcbHLH5 together with AcMYBF110 induces much greater anthocyanin accumulation in both tobacco leaves and in Actinidia arguta fruit compared with AcMYBF110 alone. Moreover, this activation is further enhanced by adding AcWDR1. We found that both AcMYBF110 and AcWDR1 interact with all three AcbHLH factors, while AcMYBF110 also interacts with AcWDR1 to form three different MBW complexes that have different regulatory roles in anthocyanin accumulation of kiwifruit. The AcMYBF110-AcbHLH1-AcWDR1 complex directly targets the promoters of anthocyanin synthetic genes. Other features of the regulatory pathways identified include promotion of AcMYBF110, AcbHLH1,and AcWDR1 activities by this MBW complex, providing for both reinforcement and feedback regulation, whereas the AcMYBF110-AcbHLH4/5-AcWDR1 complex is indirectly involved in the regulation of anthocyanin synthesis by activating the promoters of AcbHLH1 and AcWDR1 to amplify the regulation signals of the first MBW complex.

The myeloblastosis (MYB) transcription factors are essential for plant stress responses. They can enhance plant tolerance to abiotic stresses (e.g., drought, salinity, and cold) via improved physiological and biochemical responses including the accumulation of metabolites. In this study, we constructed a Panax ginseng MYB4 (PgMYB4) gene expression vector and established the stable transgenic Arabidopsis thaliana lines to study the effects of this gene on plant stress tolerance. The germination rate and seedling taproot length were greater for the PgMYB4-overexpressing plants than for the wild-type plants. Accordingly, the overexpression of PgMYB4 in Arabidopsis enhanced seedling tolerance to drought, salt, and cold conditions. Under drought stress, the relative chlorophyll content decreased less, the proline content increased more, and the water loss rate decreased more in the transgenic plants than in the wild type. The expressions of stress-related genes responsive to dehydration 19A, responsive to dehydration 22, responsive to desiccation 29A, cold-regulated 15A, cold-regulated 47, and pyrroline-5-carboxylate synthase 1 were significantly upregulated in the transgenic Arabidopsis plants. Under high salt stress, the kinesin 1 (KIN1) expression was significantly upregulated in the transgenic plants. In response to the low temperature stress, the dehydration-responsive element binding protein 2A and KIN1 expressions increased dramatically in the transgenic Arabidopsis plants. Thus, PgMYB4 positively regulated the stress tolerance gene networks, which promoted the expression of anti-stress effector genes. This gene may be useful for ginseng breeding programs aiming to develop new cultivars with enhanced stress tolerance.

KEY MESSAGE: we identified a functional chromogen gene C from wild rice, providing a new insight of anthocyanin biosynthesis pathway in indica and japonica. Accumulation of anthocyanin is a desirable trait to be selected in rice domestication, but the molecular mechanism of anthocyanin biosynthesis in rice remains largely unknown. In this study, a novel allele of chromogen gene C, OrC1, from Oryza rufipongon was cloned and identified as a determinant regulator of anthocyanin biosynthesis. Although OrC1 functions in purple apiculus, leaf sheath and stigma in indica background, it only promotes purple apiculus in japonica. Transcriptome analysis revealed that OrC1 regulates flavonoid biosynthesis pathway and activates a few bHLH and WD40 genes of ternary MYB-bHLH-WD40 complex in indica. Differentially expressed genes and metabolites were found in the indica and japonica backgrounds, indicating that OrC1 activated the anthocyanin biosynthetic genes OsCHI, OsF3H and OsANS and produced six metabolites independently. Artificial selection and domestication of C1 gene in rice occurred on the coding region in the two subspecies independently. Our results reveal the regulatory system and domestication of C1, provide new insights into MYB transcript factor involved in anthocyanin biosynthesis, and show the potential of engineering anthocyanin biosynthesis in rice.

Flavonoids are a class of commonly occurring natural compounds in the plant kingdom with various biological activities. This study compares the content of flavonoids in Cyclocarya paliurus at different developmental stages to better inform the selection of the optimal picking period. Thus, we analyzed the transcriptome and metabolome of C. paliurus at different developmental stages. The transcriptome analysis revealed 44 genes involved in the biosynthesis of flavonoids in C. paliurus, with 10 differentially expressed genes across the four different developmental stages. The metabolites were separated and identified by a combination of chromatography and mass spectrometry, followed by multi-reaction monitoring mode analysis of triple quadrupole mass spectrometry for complete metabolite quantification. In the flavonoid synthesis pathway, a total of 137 differential flavonoids were detected. The joint transcriptome and metabolome analysis showed that the expression trends in differential metabolites and genes were significantly related. Four MYB transcription factors and two bHLH transcription factors that are closely related to flavonoid biosynthesis were identified. The regulation network of flavonoid biosynthesis in C. paliurus was thus established, providing guidance for follow-up research.

Trichomes are specialized structures derived from epidermal cells. Apart from providing protection to plants, trichomes are source of medicinally important compounds. Although genes involved in initiation and patterning of trichomes have been characterized in some plants like Arabidopsis thaliana, but our knowledge about the development of these trichomes is still limited. Recent studies confirm the role of different transcription factors in trichome development of plants. In model plant Arabidopsis different transcription factor families have been studied in detail. It has been found that transcription factors like bHLH, R2R3 MYB form an activation complex with TTG1 which initiate trichome formation while as R3 MYB type transcription factors inhibit the formation of activation complex. The activation complex acts as a major trigger in the regulation of trichome development. It would be interesting to know whether such activation complex occurs in other plants like Nicotiana tobaccum, Lycopersicum esculentum, Artemisia annua etc. In this review, we focus on recent updates on transcriptional regulation of trichome development and conservation of these regulators in various species of pharmacologically and economically important plants. We have briefly discussed the cross talk of transcription factors with different hormones during trichome development. Further we have elaborated fundamental research ideologies on trichome development and translation of this research from model plant to plants of industrial interest.

Flavonoids are natural pigments occurring in plants and are present in fruits, leaves, stems, roots, and flowers. Tobacco plants transformed with an MYB regulatory gene from either Solanum chilense (Sc) or S. lycopersicum (Sl) demonstrate that ScANT1 induces a higher level of anthocyanin accumulation in comparison to SlANT1 and that this gene is sufficient to promote increased anthocyanin levels. We compared the aptitude of ScANT1 protein to induce anthocyanin accumulation to that of SlANT1 protein in tobacco plants. We also tested the effect of amino acid substitutions in ScANT1 and SlANT1. We examined these synthetic alleles' effect following the over-expression of additional anthocyanin synthesis regulators, such as the tomato bHLH (SlJAF13) protein. Our results show that the amino acid changes that differentiate ScANT1 from SlANT1 are the main contributors to the advantage that ScANT1 has over SlANT1 in anthocyanin accumulation per transcript unit. We further demonstrated that altering the amino acid composition of SlANT1 could increase anthocyanin accumulation, while reciprocally modifying ScANT1 lowers the anthocyanin level. These results confirm the increased anthocyanin level in tobacco is attributed to the amino acid differences between ScANT1 and SlANT1. We also show that the co-expression of SlJAF13 with SlANT1 in tobacco plants represses the anthocyanin production.

Freezing stress is a serious environmental factor that obstructs plant development. The root endophytic fungus Piriformospora indica has proved to be effective to confer abiotic stress tolerance to host plants. To investigate how P. indica improves freezing tolerance, we compared the expression profiles of P. indica-colonized and uncolonized Arabidopsis seedlings either exposed to freezing stress or not. Nearly 24 million (93.5%) reads were aligned on the Arabidopsis genome. 634 genes were differentially expressed between colonized and uncolonized Arabidopsis exposed to freezing stress. Interestingly, 193 Arabidopsis genes did not respond to freezing stress but were up-regulated by P. indica under freezing stress. Freezing stress-responsive genes encoded various members of the WRKY, ERF, bHLH, HSF, MYB and NAC transcription factor families. The qRT-PCR analyses confirmed the high-throughput sequencing results for 28 genes. Functional enrichment analysis indicated that the fungus mainly controls genes for freezing-stress related proteins involved in lipid and ion transport, metabolism pathways and phytohormone signaling. Our findings identified novel target genes of P. indica in freezing-stress exposed plants and highlight the benefits of the endophyte for plants exposed to a less investigated environmental threat.

Endophytic fungi generally produce secondary metabolites that have beneficial biological activities such as anticancer, antiviral, antibacterial or antioxidant compounds. The aim of this study was to examine the antioxidant activity of galling-galing leaf endophytic fungi isolate ( Cayratia trifolia L.) by 1,1-diphenyl-2-picrylhydrazil (DPPH) method. This study used IDGG-03 endophytic fungi isolate fermented for 21 days using MYB medium then extracted and evaporation was carried out. The fermentate extract obtained was then tested qualitatively and quantitatively for antioxidant activity. Qualitative results using TLC plates indicate that galling-leaf fungi isolates have antioxidant activity which is characterized by the presence of yellow spots on a purple background. And in the quantitative test IC50 68,133 µg / mL was obtained, which indicated the antioxidant activity of galling-vane leaf endophytic fungi isolate ( Cayratia trifolia L.) classified as a strong antioxidant.