Enhancing transcriptome analysis in medicinal plants: multiple unigene sets in Astragalus membranaceus.

Astragalus membranaceus is a medicinal plant mainly used in East Asia and contains abundant secondary metabolites. Despite the importance of this plant, the available genomic and genetic information is still limited. De novo transcriptome construction is recognized as an essential method for transcriptome research when reference genome information is incomplete. In this study, we constructed three individual transcriptome sets (unigene sets) for detailed analysis of the phenylpropanoid biosynthesis pathway, a major metabolite of A. membranaceus. Set-1 was a circular consensus sequence (CCS) generated using PacBio sequencing (PacBio-seq). Set-2 consisted of hybridized assembled unigenes with Illumina sequencing (Illumina-seq) reads and PacBio CCS using rnaSPAdes. Set-3 unigenes were assembled from Illumina-seq reads using the Trinity software. Construction of multiple unigene sets provides several advantages for transcriptome analysis. First, it provides an appropriate expression filtering threshold for assembly-based unigenes: a threshold transcripts per million (TPM) ≥ 5 removed more than 88% of assembly-based unigenes, which were mostly short and low-expressing unigenes. Second, assembly-based unigenes compensated for the incomplete length of PacBio CCSs: the ends of the 5`/3` untranslated regions of phenylpropanoid-related unigenes derived from set-1 were incomplete, which suggests that PacBio CCSs are unlikely to be full-length transcripts. Third, more isoform unigenes could be obtained from multiple unigene sets; isoform unigenes missing in Set-1 were detected in set-2 and set-3. Finally, gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses showed that phenylpropanoid biosynthesis and carbohydrate metabolism were highly activated in A. membranaceus roots. Various sequencing technologies and assemblers have been developed for de novo transcriptome analysis. However, no technique is perfect for de novo transcriptome analysis, suggesting the need to construct multiple unigene sets. This method enables efficient transcript filtering and detection of longer and more diverse transcripts.

Comparative analysis of mitochondrial genomes of Schisandra repanda and Kadsura japonica.

The family Schisandraceae is a basal angiosperm plant group distributed in East and Southeast Asia and includes many medicinal plant species such as Schisandra chinensis. In this study, mitochondrial genomes (mitogenomes) of two species, Schisandra repanda and Kadsura japonica, in the family were characterized through de novo assembly using sequencing data obtained with Oxford Nanopore and Illumina sequencing technologies. The mitogenomes of S. repanda were assembled into one circular contig (571,107 bp) and four linear contigs (10,898-607,430 bp), with a total of 60 genes: 38 protein-coding genes (PCGs), 19 tRNA genes, and 3 rRNA genes. The mitogenomes of K. japonica were assembled into five circular contigs (211,474-973,503 bp) and three linear contigs (8,010-72,712 bp), with a total of 66 genes: 44 PCGs, 19 tRNA genes, and 3 rRNA genes. The mitogenomes of the two species had complex structural features with high repeat numbers and chloroplast-derived sequences, as observed in other plant mitogenomes. Phylogenetic analysis based on PCGs revealed the taxonomical relationships of S. repanda and K. japonica with other species from Schisandraceae. Finally, molecular markers were developed to distinguish between S. repanda, K. japonica, and S. chinensis on the basis of InDel polymorphisms present in the mitogenomes. The mitogenomes of S. repanda and K. japonica will be valuable resources for molecular and taxonomic studies of plant species that belong to the family Schisandraceae.

Chromosome-Scale Genome Assembly and Triterpenoid Saponin Biosynthesis in Korean Bellflower (Platycodon grandiflorum).

Platycodon grandiflorum belongs to the Campanulaceae family and is an important medicinal and food plant in East Asia. However, on the whole, the genome evolution of P. grandiflorum and the molecular basis of its major biochemical pathways are poorly understood. We reported a chromosome-scale genome assembly of P. grandiflorum based on a hybrid method using Oxford Nanopore Technologies, Illumina sequences, and high-throughput chromosome conformation capture (Hi-C) analysis. The assembled genome was finalized as 574 Mb, containing 41,355 protein-coding genes, and the genome completeness was assessed as 97.6% using a Benchmarking Universal Single-Copy Orthologs analysis. The P. grandiflorum genome comprises nine pseudo-chromosomes with 56.9% repeat sequences, and the transcriptome analysis revealed an expansion of the 14 beta-amylin genes related to triterpenoid saponin biosynthesis. Our findings provide an understanding of P. grandiflorum genome evolution and enable genomic-assisted breeding for the mass production of important components such as triterpenoid saponins.

The chromosome-level genome assembly of lance asiabell (Codonopsis lanceolata), a medicinal and vegetable plant of the Campanulaceae family.

Codonopsis lanceolata (2n = 2x = 16) belongs to the Campanulaceae family and is a valuable medicinal and vegetable plant primarily found in East Asia. Several studies have demonstrated its excellent pharmacological effects, for example in bronchial treatment. However, genomic information of C. lanceolata is scarce, hindering studies on crop improvement of the species. Here, we report a high-quality chromosome-level genome assembly of C. lanceolata based on a hybrid method using Nanopore long-read, Illumina short-read, and Hi-C data. The assembled genome was completed as 1,273 Mb (84.5% of the estimated genome size), containing eight pseudo-chromosomes, ranging from 101.3 to 184.3 Mb. The genome comprised of 71.3% repeat sequences and 46,005 protein-coding genes, of which 85.7% genes were functionally annotated. Completeness of the assembled genome and genes was assessed to be 97.5% and 90.4%, respectively, by Benchmarking Universal Single-Copy Orthologs analysis. Phylogenetic and synteny analysis revealed that C. lanceolata was closely related to Platycodon grandiflorus in the Campanulaceae family. Gene family evolution revealed significant expansion of related genes involved in saponin biosynthesis in the C. lanceolata genome. This is the first reference genome reported for C. lanceolata. The genomic data produced in this study will provide essential information for further research to improve this medicinal plant and will broaden the understanding of the Campanulaceae family.

First Contiguous Genome Assembly of Japanese Lady Bell (Adenophora triphylla) and Insights into Development of Different Leaf Types.

Adenophora triphylla is an important medicinal and food plant found in East Asia. This plant is rich in secondary metabolites such as triterpenoid saponin, and its leaves can develop into different types, such as round and linear, depending on the origin of germination even within the same species. Despite this, few studies have comprehensively characterized the development processes of different leaf types and triterpenoid saponin pathways in this plant. Herein, we provide the first report of a high-quality genome assembly of A. triphylla based on a combination of Oxford Nanopore Technologies and Illumina sequencing methods. Its genome size was estimated to be 2.6 Gb, and the assembled genome finalized as 2.48 Gb, containing 57,729 protein-coding genes. Genome completeness was assessed as 95.6% using the Benchmarking Universal Single-Copy Orthologs score. The evolutionary divergence of A. triphylla was investigated using the genomes of five plant species, including two other species in the Campanulaceae family. The species A. triphylla diverged approximately 51-118 million years ago from the other four plants, and 579 expanded/contracted gene families were clustered in the Gene Ontology terms. The expansion of the ß-amyrin synthase (bAS) gene, a key enzyme in the triterpenoid saponin pathway, was identified in the A. triphylla genome. Furthermore, transcriptome analysis of the two leaf types revealed differences in the activity of starch, sucrose, unsaturated fatty acid pathways, and oxidoreductase enzymes. The heat and endoplasmic reticulum pathways related to plant stress were active in the development of round type leaf, while an enhancement of pyrimidine metabolism related to cell development was confirmed in the development of the linear type leaf. This study provides insight into the evolution of bAS genes and the development of different leaf types in A. triphylla.

Co-Expression Analysis Reveals Differential Expression of Homologous Genes Associated with Specific Terpenoid Biosynthesis in Rehmannia glutinosa.

Terpenoids are naturally occurring compounds involved in respiration, photosynthesis, membrane fluidity, and pathogen interactions and are classified according to the structure of their carbon skeleton. Although most terpenoids possess pharmacological activity, knowledge about terpenoid metabolism in medicinal plants is insufficient. Rehmannia glutinosa (R. glutinosa) is a traditional herb that is widely used in East Asia and has been reported to contain various terpenoids. In this study, we performed a comprehensive transcriptome analysis of terpenoid metabolism in R. glutinosa using two RNA sequencing platforms: Illumina and PacBio. The results show that the sterol, saponin, iridoid, and carotenoid pathways are active in R. glutinosa. Sterol and saponin biosynthesis were mevalonate pathway dependent, whereas iridoid and carotenoid biosynthesis were methylerythritol 4-phosphate pathway dependent. In addition, we found that the homologous genes of key enzymes involved in terpenoid metabolism were expressed differentially and that the differential expression of these genes was associated with specific terpenoid biosynthesis. The different expression of homologous genes encoding acetyl-CoA acetyltransferase, 3-hydroxy-3-methylglutaryl-CoA reductase, mevalonate kinase, mevalonate diphosphate decarboxylase, farnesyl pyrophosphate synthase, squalene synthase, and squalene epoxidase was associated with sterol and saponin biosynthesis. Homologous genes encoding 1-deoxy-D-xylulose 5-phosphate synthase were also differentially expressed and were associated with carotenoid and iridoid biosynthesis. These results suggest that the biosynthesis of specific terpenoids can be regulated by the homologous of key enzymes involved in plant terpenoid metabolism.

Long-read transcriptome sequencing provides insight into lignan biosynthesis during fruit development in Schisandra chinensis.

BACKGROUND: Schisandra chinensis, an ancient member of the most basal angiosperm lineage which is known as the ANITA, is a fruit-bearing vine with the pharmacological effects of a multidrug system, such as antioxidant, anti-inflammatory, cardioprotective, neuroprotective, anti-osteoporosis effects. Its major bioactive compound is represented by lignans such as schisandrin. Molecular characterization of lignan biosynthesis in S. chinensis is of great importance for improving the production of this class of active compound. However, the biosynthetic mechanism of schisandrin remains largely unknown. RESULTS: To understand the potential key catalytic steps and their regulation of schisandrin biosynthesis, we generated genome-wide transcriptome data from three different tissues of S. chinensis cultivar Cheongsoon, including leaf, root, and fruit, via long- and short-read sequencing technologies. A total of 132,856 assembled transcripts were generated with an average length of 1.9 kb and high assembly completeness. Overall, our data presented effective, accurate gene annotation in the prediction of functional pathways. In particular, the annotation revealed the abundance of transcripts related to phenylpropanoid biosynthesis. Remarkably, transcriptome profiling during fruit development of S. chinensis cultivar Cheongsoon revealed that the phenylpropanoid biosynthetic pathway, specific to coniferyl alcohol biosynthesis, showed a tendency to be upregulated at the postfruit development stage. Further the analysis also revealed that the pathway forms a transcriptional network with fruit ripening-related genes, especially the ABA signaling-related pathway. Finally, candidate unigenes homologous to isoeugenol synthase 1 (IGS1) and dirigent-like protein (DIR), which are subsequently activated by phenylpropanoid biosynthesis and thus catalyze key upstream steps in schisandrin biosynthesis, were identified. Their expression was increased at the postfruit development stage, suggesting that they may be involved in the regulation of schisandrin biosynthesis in S. chinensis. CONCLUSIONS: Our results provide new insights into the production and accumulation of schisandrin in S. chinensis berries and will be utilized as a valuable transcriptomic resource for improving the schisandrin content.

Analysis of Phenotypic Characteristics and Sucrose Metabolism in the Roots of Raphanus sativus L.

The taproot of radish (Raphanus sativus L.) is an important sink organ; it is morphologically diverse and contains large amounts of secondary metabolites. Sucrose metabolism is believed to be important in the development of sink organs. We measured the amounts of glucose, fructose, and sucrose in the roots of sixty three radish accessions and analyzed the association between the sugar content and the root phenotype. Fructose content correlated with the root color and length characteristics, glucose was the most abundant sugar in the roots, and the sucrose content was very low, compared to that of the hexoses in most of the accessions. Expression analysis of the genes involved in sucrose metabolism, transportation, starch synthesis, and cell wall synthesis was performed through RNA sequencing. The genes encoding sucrose synthases (SUSY) and the enzymes involved in the synthesis of cellulose were highly expressed, indicating that SUSY is involved in cell wall synthesis in radish roots. The positive correlation coefficient (R) between the sucrose content and the expression of cell wall invertase and sugar transporter proteins suggest that hexose accumulation could occur through the apoplastic pathway in radish roots. A positive R score was also obtained when comparing the expression of genes encoding SUSY and fructokinase (FK), suggesting that the fructose produced by SUSY is mostly phosphorylated by FK. In addition, we concluded that sucrose was the most metabolized sugar in radish roots.

Systemic Expression of Genes Involved in the Plant Defense Response Induced by Wounding in Senna tora.

Wounds in tissues provide a pathway of entry for pathogenic fungi and bacteria in plants. Plants respond to wounding by regulating the expression of genes involved in their defense mechanisms. To analyze this response, we investigated the defense-related genes induced by wounding in the leaves of Senna tora using RNA sequencing. The genes involved in jasmonate and ethylene biosynthesis were strongly induced by wounding, as were a large number of genes encoding transcription factors such as ERFs, WRKYs, MYBs, bHLHs, and NACs. Wounding induced the expression of genes encoding pathogenesis-related (PR) proteins, such as PR-1, chitinase, thaumatin-like protein, cysteine proteinase inhibitor, PR-10, and plant defensin. Furthermore, wounding led to the induction of genes involved in flavonoid biosynthesis and the accumulation of kaempferol and quercetin in S. tora leaves. All these genes were expressed systemically in leaves distant from the wound site. These results demonstrate that mechanical wounding can lead to a systemic defense response in the Caesalpinioideae, a subfamily of the Leguminosae. In addition, a co-expression analysis of genes induced by wounding provides important information about the interactions between genes involved in plant defense responses.

Induction of Glucoraphasatin Biosynthesis Genes by MYB29 in Radish (Raphanus sativus L.) Roots.

Glucoraphasatin (GRH) is a specific aliphatic glucosinolate (GSL) that is only abundant in radish (Raphanus sativus L.). The gene expression regulating GRH biosynthesis in radish is still poorly understood. We employed a total of 59 radish accessions to analyze GSL profiles and showed that GRH was specific and predominant among the aliphatic GSLs in radish roots. We selected five accessions roots with high, moderate and low GSL biosynthesis, respectively, to conduct a comparative transcriptome analysis and the qRT-PCR of the biosynthesis genes for aliphatic GSLs. In this study, among all the accessions tested, roots with the accession RA157-74 had a high GRH content and showed a significant expression of the aliphatic GSL biosynthesis genes. We defined the genes involved in the GRH biosynthesis process and found that they were regulated by a transcription factor (RSG00789) at the MYB29 locus in radish roots. We found 13 aliphatic GSL biosynthesis genes regulated by the RSG00789 gene in the GRH biosynthesis pathway.