Comparative Chemical and Transcriptome Analysis of Four Brazilian Piper Species During Ontogeny

Comparative Chemical and Transcriptome Analysis of Four Brazilian
Piper Species During Ontogeny

Dimitre A. Ivanov1*, Paula Elbl2, Diego T. de Souza2, Lydia F. Yamaguchi1,
Eny I.S. Floh2, Massuo J. Kato1

1Laboratory of Natural Product Chemistry, Institute of Chemistry, University of São Paulo
Av. Prof. Lineu Prestes, 748 Butantã 05508-000 São Paulo-SP, Brazil
2Institute of Biosciences, University of São Paulo
Rua do Matão, 277, 05508-090, Cidade Universitária - São Paulo-SP, Brazil
*[email protected]

Piper is a genus of Pantropical distribution with economic and ecological importance within the family Piperaceae. In this study, the chemical differentiation of four Piper species was examined during ontogeny in order to complement previous phytochemical studies that determined the extensive chemical diversity of Piper species. These phytochemical studies, however, mainly focused on the analysis of mature plants, due to the large amount of biomass required for chemical analyses during bioprospecting. The lack of an in-depth examination of the secondary chemistry of Piper seedlings, despite their important ecological role in the establishment of plant communities (Kato et al. 1992) and previous reports of the existence of species-specific chemical variability between seedling and mature Piper plants (Gaia et al. 2014) were the main drivers for the completion of this study.

As part of this analysis, the chemical composition and transcriptome of Piper arboreum Aubl., Piper gaudichaudianum Kunth ex Steud., Piper malacophyllum (C. Presl) C. DC and Piper tuberculatum Jacq. at two different life stages (2-month old seedlings and mature adult plants) were examined using HPLC-DAD-MS and RNA-seq, respectively. This data was then used to attempt to discern the regulatory steps involved in the formation of phenylpropanoid compounds and other metabolites associated with the major secondary metabolic pathways in these plants.

The seedling and adult leaf transcriptomes of P arboreum, P. gaudichaudianum, P. malacophyllum and P. tuberculatum were assembled using a total of 164 Gb of sequencing data obtained from the Life Sciences Core Facility (LaCTAD) from the State University of Campinas (UNICAMP). The number of 100 bp paired-end reads obtained per sample varied from ~16 million to ~190 million depending on the species and treatment. The data obtained was pre-processed by using the program FastQC to evaluate the quality of the reads obtained after which reads were trimmed using the program SeqyClean. Read quality was then improved through the removal of contaminating foreign sequences with the program HISAT2, by utilizing a 17.6 Gb decontamination database compiled from 1016 organisms. Following the quality trimming and decontamination of the sequencing libraries obtained, assembly of all of the recovered high quality paired-end reads was performed using the Trinity v.2.4.0 software package (Grabherr et al. 2011). In total, 483836 Trinity “genes” were assembled and the number of trinity transcripts recovered was 1123734. These Trinity “genes” were then annotated using the Trinotate platform for automatic functional annotation of transcriptomes (Haas et al. 2013) which utilizes the databases of Uniprot, Pfam, Gene Ontology and Kegg Orthology. This functional annotation subsequently allowed for the identification of differentially expressed genes (DEGs) between the two life stages of each Piper species studied.

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Grabherr, M. G. et al. 2011. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nature Biotechnology. 29; 644-652.
Haas B.J. et al. (2013) De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis. Nature Protocols 8; 1494–1512.
Kato, M.J et al. 1992. Flavones and lignans in flowers, fruits and seedlings of Virola venosa. Phytochemistry. 31; 283-287.