AtPAP1 Interacts With and Activates SmbHLH51, a Positive Regulator to Phenolic Acids Biosynthesis in Salvia miltiorrhiza

Phenolic acids from Salvia miltiorrhiza have drawn considerable attention in recent years because of their remarkable pharmacological activities. We previously reported that Arabidopsis thaliana transcription factor production of anthocyanin pigment 1 (AtPAP1) has strong capability to promote the production of phenolic acids in S. miltiorrhiza. However, the responsible molecular mechanism is unclear. Here, we analyzed the transcriptome of transgenic S. miltiorrhiza that over-expressed AtPAP1. Transcriptome analysis revealed 4,152 genes that were differentially expressed due to ectopic AtPAP1 overexpression. SmbHLH51, a novel bHLH gene significantly up-regulated by constitutive expression of AtPAP1, was isolated from S. miltiorrhiza for detailed functional characterization. SmbHLH51 localizes in the nuclei and interacts with AtPAP1, indicating that they probably comprise a regulatory transcription complex. Enhanced or reduced expression of SmbHLH51 was achieved in S. miltiorrhiza by gain- or loss-of-function assays, respectively, revealing that SmbHLH51 is a positive transcriptional regulator of the pathway for phenolic acid biosynthesis. We propose that applying this functional genomics approach through the transcriptomic analyses is an efficient means for identifying novel genes involved in plant secondary metabolism

Genome-Wide Comprehensive Analysis the Molecular Phylogenetic Evaluation and Tissue-Specific Expression of SABATH Gene Family in Salvia miltiorrhiza

The plant SABATH gene family is a group of O-methyltransferases (O-MTs), which belongs to the S-adenosyl-l-methionine-dependent methyltransferases (SAM-MTs). The resulting reaction products of SABATH genes play an important role in various processes of plant development. In this study, a total of 30 SABATH genes were detected in Salvia miltiorrhiza, which is an important medicinal plant, widely used to treat cardiovascular disease. Multiple sequence alignment and phylogenetic analyses showed that SmSABATH genes could be classified into three groups. The ratios of non-synonymous (Ka) and synonymous (Ks) substitution rates of 11 pairs paralogous of SmSABATH genes revealed that the SmSABATH genes had gone through purifying selection. Positive selection analyses using site models and branch-site models indicated that SmSABATH genes had undergone selective pressure for adaptive evolution. Functional divergence analyses suggested that the SmSABATH subgroup genes were divergent in terms of functions and positive selection sites that contributed to a functional divergence among the subgroups that were detected. Tissue-specific expression showed that the SABATH gene family in S. miltiorrhiza was primarily expressed in stems and leaves

Selection and Validation of Appropriate Reference Genes for qRT-PCR Analysis in Isatis indigotica Fort.

Due to its sensitivity and specificity, real-time quantitative PCR (qRT-PCR) is a popular technique for investigating gene expression levels in plants. Based on the Minimum Information for Publication of Real-Time Quantitative PCR Experiments (MIQE) guidelines, it is necessary to select and validate putative appropriate reference genes for qRT-PCR normalization. In the current study, three algorithms, geNorm, NormFinder, and BestKeeper, were applied to assess the expression stability of 10 candidate reference genes across five different tissues and three different abiotic stresses in Isatis indigotica Fort. Additionally, the IiYUC6 gene associated with IAA biosynthesis was applied to validate the candidate reference genes. The analysis results of the geNorm, NormFinder, and BestKeeper algorithms indicated certain differences for the different sample sets and different experiment conditions. Considering all of the algorithms, PP2A-4 and TUB4 were recommended as the most stable reference genes for total and different tissue samples, respectively. Moreover, RPL15 and PP2A-4 were considered to be the most suitable reference genes for abiotic stress treatments. The obtained experimental results might contribute to improved accuracy and credibility for the expression levels of target genes by qRT-PCR normalization in I. indigotica

The complete chloroplast genome sequence of <i>Epipremnum aureum</i> and its comparative analysis among eight Araceae species

<div><p><i>Epipremnum aureum</i> is an important foliage plant in the Araceae family. In this study, we have sequenced the complete chloroplast genome of <i>E</i>. <i>aureum</i> by using Illumina Hiseq sequencing platforms. This genome is a double-stranded circular DNA sequence of 164,831 bp that contains 35.8% GC. The two inverted repeats (IRa and IRb; 26,606 bp) are spaced by a small single-copy region (22,868 bp) and a large single-copy region (88,751 bp). The chloroplast genome has 131 (113 unique) functional genes, including 86 (79 unique) protein-coding genes, 37 (30 unique) tRNA genes, and eight (four unique) rRNA genes. Tandem repeats comprise the majority of the 43 long repetitive sequences. In addition, 111 simple sequence repeats are present, with mononucleotides being the most common type and di- and tetranucleotides being infrequent events. Positive selection pressure on <i>rps12</i> in the <i>E</i>. <i>aureum</i> chloroplast has been demonstrated via synonymous and nonsynonymous substitution rates and selection pressure sites analyses. <i>Ycf15</i> and <i>infA</i> are pseudogenes in this species. We constructed a Maximum Likelihood phylogenetic tree based on the complete chloroplast genomes of 38 species from 13 families. Those results strongly indicated that <i>E</i>. <i>aureum</i> is positioned as the sister of <i>Colocasia esculenta</i> within the Araceae family. This work may provide information for further study of the molecular phylogenetic relationships within Araceae, as well as molecular markers and breeding novel varieties by chloroplast genetic-transformation of <i>E</i>. <i>aureum</i> in particular.</p></div

De Novo Assembly and Analysis of Polygonatum sibiricum Transcriptome and Identification of Genes Involved in Polysaccharide Biosynthesis

Polygonatum sibiricum polysaccharides (PSPs) are used to improve immunity, alleviate dryness, promote the secretion of fluids, and quench thirst. However, the PSP biosynthetic pathway is largely unknown. Understanding the genetic background will help delineate that pathway at the molecular level so that researchers can develop better conservation strategies. After comparing the PSP contents among several different P. sibiricum germplasms, we selected two groups with the largest contrasts in contents and subjected them to HiSeq2500 transcriptome sequencing to identify the candidate genes involved in PSP biosynthesis. In all, 20 kinds of enzyme-encoding genes were related to PSP biosynthesis. The polysaccharide content was positively correlated with the expression patterns of β-fructofuranosidase (sacA), fructokinase (scrK), UDP-glucose 4-epimerase (GALE), Mannose-1-phosphate guanylyltransferase (GMPP), and UDP-glucose 6-dehydrogenase (UGDH), but negatively correlated with the expression of Hexokinase (HK). Through qRT-PCR validation and comprehensive analysis, we determined that sacA, HK, and GMPP are key genes for enzymes within the PSP metabolic pathway in P. sibiricum. Our results provide a public transcriptome dataset for this species and an outline of pathways for the production of polysaccharides in medicinal plants. They also present more information about the PSP biosynthesis pathway at the molecular level in P. sibiricum and lay the foundation for subsequent research of gene functions

Genome-Wide Identification of the <i>Hypericum perforatum</i> WRKY Gene Family Implicates <i>HpWRKY85</i> in Drought Resistance

WRKY, named for its special heptapeptide conserved sequence WRKYGOK, is one of the largest transcription factor families in plants and is widely involved in plant responses to biotic, abiotic, and hormonal stresses, especially the important regulatory function in response to drought stress. However, there is no complete comprehensive analysis of this family in H. perforatum, which is one of the most extensively studied plants and is probably the best-known herbal medicine on the market today, serving as an antidepressant, neuroprotective, an antineuralgic, and an antiviral. Here, we identified 86 HpWRKY genes according to the whole genome database of H. perforatum, and classified them into three groups through phylogenetic analysis. Gene structure, conserved domain, motif, cis-elements, gene ontology, and expression profiling were performed. Furthermore, it was found that HpWRKY85, a homologous gene of AtWRKY75, showed obvious responses to drought treatment. Subcellular localization analysis indicated that this protein was localized in the nucleus by the Arabidopsis protoplasts transient transfection. Meanwhile, HpWRKY85-overexpressing Arabidopsis plants showed a stronger ability of root growth and scavenging endogenous reactive oxygen species. The results provide a reference for further understanding the role of HpWRKY85 in the molecular mechanism of drought resistance of H. perforatum

Overexpression of SmMYC2 Increases the Production of Phenolic Acids in Salvia miltiorrhiza

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Systematic Identification and Functional Analysis of the <i>Hypericum perforatum</i> L. bZIP Gene Family Indicating That Overexpressed <i>HpbZIP69</i> Enhances Drought Resistance

Basic leucine zipper (bZIP) transcription factors play significant roles in plants’ growth and development processes, as well as in response to biological and abiotic stresses. Hypericum perforatum is one of the world’s top three best-selling herbal medicines, mainly used to treat depression. However, there has been no systematic identification or functional analysis of the bZIP gene family in H. perforatum. In this study, 79 HpbZIP genes were identified. Based on phylogenetic analysis, the HpbZIP gene family was divided into ten groups, designated A–I and S. The physicochemical properties, gene structures, protein conserved motifs, and Gene Ontology enrichments of all HpbZIPs were systematically analyzed. The expression patterns of all genes in different tissues of H. perforatum (i.e., root, stem, leaf, and flower) were analyzed by qRT-PCR, revealing the different expression patterns of HpbZIP under abiotic stresses. The HpbZIP69 protein is localized in the nucleus. According to the results of the yeast one-hybrid (Y1H) assays, HpbZIP69 can bind to the HpASMT2 (N-acetylserotonin O-methyltransferase) gene promoter (G-box cis-element) to activate its activity. Overexpressing HpbZIP69 in Arabidopsis wild-type lines enhanced their tolerance to drought. The MDA and H2O2 contents were significantly decreased, and the activity of superoxide dismutase (SOD) was considerably increased under the drought stress. These results may aid in additional functional studies of HpbZIP transcription factors, and in cultivating drought-resistant medicinal plants