Michimi Nakamura

Heavy metal tolerance of transgenic tobacco plants over-expressing cysteine synthase.

Cysteine synthase [O-acetyl-l-serine(thiol)lyase] catalyzes the final step for l-cysteine biosynthesis in plants. The tolerance of transgenic tobacco plants over-expressing cysteine synthase cDNA in cytosol (3F), chloroplasts (4F) and in both organelles (F1) was investigated towards heavy metals such as Cd, Se, Ni, Pb and Cu. The transgenic plants were significantly more tolerant than wild-type plants in agar medium containing Cd, Se and Ni. The F1 transgenic plants had a higher resistance than other transgenic lines towards these metals and could enhance accumulation of Cd in shoot. These results suggest that the transgenic plants over-expressing cysteine synthase both in cytosol and chloroplasts can be applicable to phyto-remediation of Cd from contaminated soils.

RNA-seq Transcriptome Analysis of Panax japonicus, and Its Comparison with Other Panax Species to Identify Potential Genes Involved in the Saponins Biosynthesis.

The Panax genus has been a source of natural medicine, benefitting human health over the ages, among which the Panax japonicus represents an important species. Our understanding of several key pathways and enzymes involved in the biosynthesis of ginsenosides, a pharmacologically active class of metabolites and a major chemical constituents of the rhizome extracts from the Panax species, are limited. Limited genomic information, and lack of studies on comparative transcriptomics across the Panax species have restricted our understanding of the biosynthetic mechanisms of these and many other important classes of phytochemicals. Herein, we describe Illumina based RNA sequencing analysis to characterize the transcriptome and expression profiles of genes expressed in the five tissues of P. japonicus, and its comparison with other Panax species. RNA sequencing and de novo transcriptome assembly for P. japonicus resulted in a total of 135,235 unigenes with 78,794 (58.24%) unigenes being annotated using NCBI-nr database. Transcriptome profiling, and gene ontology enrichment analysis for five tissues of P. japonicus showed that although overall processes were evenly conserved across all tissues. However, each tissue was characterized by several unique unigenes with the leaves showing the most unique unigenes among the tissues studied. A comparative analysis of the P. japonicus transcriptome assembly with publically available transcripts from other Panax species, namely, P. ginseng, P. notoginseng, and P. quinquefolius also displayed high sequence similarity across all Panax species, with P. japonicus showing highest similarity with P. ginseng. Annotation of P. japonicus transcriptome resulted in the identification of putative genes encoding all enzymes from the triterpene backbone biosynthetic pathways, and identified 24 and 48 unigenes annotated as cytochrome P450 (CYP) and glycosyltransferases (GT), respectively. These CYPs and GTs annotated unigenes were conserved across all Panax species and co-expressed with other the transcripts involved in the triterpenoid backbone biosynthesis pathways. Unigenes identified in this study represent strong candidates for being involved in the triterpenoid saponins biosynthesis, and can serve as a basis for future validation studies.

High-Throughput Sequencing and De Novo Assembly of Red and Green Forms of the Perilla frutescens var. crispa Transcriptome

Perilla frutescens var. crispa (Labiatae) has two chemo-varietal forms, i.e. red and green forms of perilla, that differ in the production of anthocyanins. To facilitate molecular biological and biochemical studies in perilla-specialized metabolism we used Illumina RNA-sequencing technology in our comprehensive comparison of the transcriptome map of the leaves of red and green forms of perilla. Sequencing generated over 1.2 billion short reads with an average length of 101 nt. De novo transcriptome assembly yielded 47,788 and 47,840 unigenes in the red and green forms of perilla plants, respectively. Comparison of the assembled unigenes and existing perilla cDNA sequences showed highly reliable alignment. All unigenes were annotated with gene ontology (GO) and Enzyme Commission numbers and entered into the Kyoto Encyclopedia of Genes and Genomes. We identified 68 differentially expressed genes (DEGs) in red and green forms of perilla. GO enrichment analysis of the DEGs showed that genes involved in the anthocyanin metabolic process were enriched. Differential expression analysis revealed that the transcript level of anthocyanin biosynthetic unigenes encoding flavonoid 3’-hydroxylase, dihydroflavonol 4-reductase, and anthocyanidin synthase was significantly higher in red perilla, while the transcript level of unigenes encoding limonene synthase was significantly higher in green perilla. Our data serve as a basis for future research on perilla bio-engineering and provide a shortcut for the characterization of new functional genes in P. frutescens.

High-throughput sequencing and de novo transcriptome assembly of Swertia japonica to identify genes involved in the biosynthesis of therapeutic metabolites

Key messageHere, we report potential transcripts involved in the biosynthesis of therapeutic metabolites inSwertia japonica, the first report of transcriptome assembly, and characterization of the medicinal plant fromSwertiagenus.AbstractSwertia genus, representing over 170 plant species including herbs such as S. chirata, S. hookeri, S. longifolia, S. japonica, among others, have been used as the traditional medicine in China, India, Korea, and Japan for thousands of years. Due to the lack of genomic and transcriptomic resources, little is known about the molecular basis involved in the biosynthesis of characteristic key bioactive metabolites. Here, we performed deep-transcriptome sequencing for the aerial tissues and the roots of S. japonica, generating over 2 billion raw reads with an average length of 101 bps. Using a combined approach of three popular assemblers, de novo transcriptome assembly for S. japonica was obtained, yielding 81,729 unigenes having an average length of 884 bps and N50 value of 1452 bps, of which 46,963 unigenes were annotated based on the sequence similarity against NCBI-nr protein database. Annotation of transcriptome assembly resulted in the identification of putative genes encoding all enzymes from the key therapeutic metabolite biosynthesis pathways. Transcript abundance analysis, gene ontology enrichment analysis, and KEGG pathway enrichment analysis revealed metabolic processes being up-regulated in the aerial tissues with respect to the roots of S. japonica. We also identified 37 unigenes as potential candidates involved in the glycosylation of bioactive metabolites. Being the first report of transcriptome assembly and annotation for any of the Swertia species, this study will be a valuable resource for future investigations on the biosynthetic pathways of therapeutic metabolites and their regulations.

Transcriptomic landscape of Pueraria lobata demonstrates potential for phytochemical study

Pueraria lobata (Willd.) Ohwi has a long and broad application in the treatment of disease. However, in the US and EU, it is treated as a notorious weed. The information to be gained from decoding the deep transcriptome profile would facilitate further research on P. lobata. In this study, more than 93 million fastq format reads were generated by Illumina’s next-generation sequencing approach using five types of P. lobata tissue, followed by CLC de novo assembly methods, ultimately yielding about 83,041 contigs in total. Then BLASTx similarity searches against the NCBI NR database and UniProtKB database were conducted. Once the duplicates among BLASTx hits were eliminated, ID mapping against the UniProt database was conducted online to retrieve Gene Ontology information. In search of the putative genes relevant to essential biosynthesis pathways, all 1,348 unique enzyme commission numbers were used to map pathways against the Kyoto Encyclopedia of Genes and Genomes. Enzymes related to the isoflavonoid and flavonoid biosynthesis pathways were focused for detailed investigation and subsequently, quantitative real-time reverse transcription polymerase chain reaction was conducted for biological validation. Metabolites of interest, puerarin and daidzin were studied by HPLC. The findings in this report may serve as a footstone for further research into this promising medicinal plant.

De Novo Deep Transcriptome Analysis of Medicinal Plants for Gene Discovery in Biosynthesis of Plant Natural Products.

Study on transcriptome, the entire pool of transcripts in an organism or single cells at certain physiological or pathological stage, is indispensable in unraveling the connection and regulation between DNA and protein. Before the advent of deep sequencing, microarray was the main approach to handle transcripts. Despite obvious shortcomings, including limited dynamic range and difficulties to compare the results from distinct experiments, microarray was widely applied. During the past decade, next-generation sequencing (NGS) has revolutionized our understanding of genomics in a fast, high-throughput, cost-effective, and tractable manner. By adopting NGS, efficiency and fruitful outcomes concerning the efforts to elucidate genes responsible for producing active compounds in medicinal plants were profoundly enhanced. The whole process involves steps, from the plant material sampling, to cDNA library preparation, to deep sequencing, and then bioinformatics takes over to assemble enormous-yet fragmentary-data from which to comb and extract information. The unprecedentedly rapid development of such technologies provides so many choices to facilitate the task, which can cause confusion when choosing the suitable methodology for specific purposes. Here, we review the general approaches for deep transcriptome analysis and then focus on their application in discovering biosynthetic pathways of medicinal plants that produce important secondary metabolites.

Comparative Characterization of the Leaf Tissue of Physalis alkekengi and Physalis peruviana Using RNA-seq and Metabolite Profiling

The genus Physalis in the Solanaceae family contains several species of benefit to humans. Examples include P. alkekengi (Chinese-lantern plant, hôzuki in Japanese) used for medicinal and for decorative purposes, and P. peruviana, also known as Cape gooseberry, which bears an edible, vitamin-rich fruit. Members of the Physalis genus are a valuable resource for phytochemicals needed for the development of medicines and functional foods. To fully utilize the potential of these phytochemicals we need to understand their biosynthesis, and for this we need genomic data, especially comprehensive transcriptome datasets for gene discovery. We report the de novo assembly of the transcriptome from leaves of P. alkekengi and P. peruviana using Illumina RNA-seq technologies. We identified 75,221 unigenes in P. alkekengi and 54,513 in P. peruviana. All unigenes were annotated with gene ontology (GO), Enzyme Commission (EC) numbers, and pathway information from the Kyoto Encyclopedia of Genes and Genomes (KEGG). We classified unigenes encoding enzyme candidates putatively involved in the secondary metabolism and identified more than one unigenes for each step in terpenoid backbone- and steroid biosynthesis in P. alkekengi and P. peruviana. To measure the variability of the withanolides including physalins and provide insights into their chemical diversity in Physalis, we also analyzed the metabolite content in leaves of P. alkekengi and P. peruviana at five different developmental stages by liquid chromatography-mass spectrometry. We discuss that comprehensive transcriptome approaches within a family can yield a clue for gene discovery in Physalis and provide insights into their complex chemical diversity. The transcriptome information we submit here will serve as an important public resource for further studies of the specialized metabolism of Physalis species.

Comparative transcriptome analyses of three medicinal Forsythia species and prediction of candidate genes involved in secondary metabolisms

The three Forsythia species, F. suspensa, F. viridissima and F. koreana, have been used as herbal medicines in China, Japan and Korea for centuries and they are known to be rich sources of numerous pharmaceutical metabolites, forsythin, forsythoside A, arctigenin, rutin and other phenolic compounds. In this study, de novo transcriptome sequencing and assembly was performed on these species. Using leaf and flower tissues of F. suspensa, F. viridissima and F. koreana, 1.28–2.45-Gbp sequences of Illumina based pair-end reads were obtained and assembled into 81,913, 88,491 and 69,458 unigenes, respectively. Classification of the annotated unigenes in gene ontology terms and KEGG pathways was used to compare the transcriptome of three Forsythia species. The expression analysis of orthologous genes across all three species showed the expression in leaf tissues being highly correlated. The candidate genes presumably involved in the biosynthetic pathway of lignans and phenylethanoid glycosides were screened as co-expressed genes. They express highly in the leaves of F. viridissima and F. koreana. Furthermore, the three unigenes annotated as acyltransferase were predicted to be associated with the biosynthesis of acteoside and forsythoside A from the expression pattern and phylogenetic analysis. This study is the first report on comparative transcriptome analyses of medicinally important Forsythia genus and will serve as an important resource to facilitate further studies on biosynthesis and regulation of therapeutic compounds in Forsythia species.