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.

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.

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.

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.

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.

Ji-Nam Kang, Gyeong-Hui Lee, Jin Yu, Mi-Hwa Choi, Simyung Lee. Plant Breed. Biotech. 2023;11:130-40. https://doi.org/10.9787/PBB.2023.11.2.130