Alkaloid production and response to natural adverse conditions in Peganum harmala: in silico transcriptome analyses

Abstract

Peganum harmala is a valuable wild plant that grows and survives under adverse conditions and produces pharmaceutical alkaloid metabolites. Using different assemblers to develop a transcriptome improves the quality of assembled transcriptome. In this study, a concrete and accurate method for detecting stress-responsive transcripts by comparing stress-related gene ontology (GO) terms and public domains was designed. An integrated transcriptome for P. harmala including 42 656 coding sequences was created by merging de novo assembled transcriptomes. Around 35 000 transcripts were annotated with more than 90% resemblance to three closely related species of Citrus, which confirmed the robustness of the assembled transcriptome; 4853 stress-responsive transcripts were identified. CYP82 involved in alkaloid biosynthesis showed a higher number of transcripts in P. harmala than in other plants, indicating its diverse alkaloid biosynthesis attributes. Transcription factors (TFs) and regulatory elements with 3887 transcripts comprised 9% of the transcriptome. Among the TFs of the integrated transcriptome, cystein2/histidine2 (C2H2) and WD40 repeat families were the most abundant. The Kyoto Encyclopedia of Genes and Genomes (KEGG) MAPK (mitogen-activated protein kinase) signaling map and the plant hormone signal transduction map showed the highest assigned genes to these pathways, suggesting their potential stress resistance. The P. harmala whole-transcriptome survey provides important resources and paves the way for functional and comparative genomic studies on this plant to discover stress-tolerance-related markers and response mechanisms in stress physiology, phytochemistry, ecology, biodiversity, and evolution. P. harmala can be a potential model for studying adverse environmental cues and metabolite biosynthesis and a major source for the production of various alkaloids.

Keywords: RNA-Seq; SSR; halophyte; model plant; transcription factor.

Fig. 1

GC content distribution of transcripts of the Peganum harmala transcriptome; the GC content of P. harmala is similar to that of Pistacia vera and Citrus, which are its close species

Fig. 2

The e-value distribution for the blast hits of comparison between the transcripts of Peganum harmala transcriptome and the NCBI NR database; it is an indicator for good coherent similarity between the query and the subject; a low e-value shows better match between the query and the subject

Fig. 3

Similarity distribution among blast hits between the transcriptome of Peganum harmala and the NCBI NR database; the higher number of hits for more positive/alignment length indicates that the query and the subject are more similar, and hence, the generated transcriptome was well structured

Fig. 4

Species distribution of blast-based comparison between the transcripts of Peganum harmala transcriptome and the NCBI NT database; the top species belong to Citrus, which is very close to Peganum and Pistacia of the same order

Fig. 5

Top GO terms with more than 3000 transcripts and their distribution among all annotated transcripts of the Peganum harmala transcriptome against the Plants database of the GO slimviewer tool; for the BP group, cellular process, metabolic process, and biosynthetic process were the top GO terms; for the CC group, cell, intracellular, and cytoplasm were the top GO terms; for the MF group, binding, protein binding, and catalytic activity were the top GO terms; BP – biological process; CC – cellular component; MF – molecular function

Fig. 6

The phylogenetic tree for species of Sapindales, Malvales, and Brassicale; Pistacia vera is the closest species to Peganum harmala by 1 tree scale distance, followed by Citrus species; alkaloid plants including Papaver somniferum, Catharanthus roseus, Nicotiana tabacum, and Eschscholzia californica are not close species to P. harmala ; Arabidopsis species are closer to P. harmala than alkaloid-producing plants

Fig. 7

Comparisons between the transcripts of Peganum harmala and the data of other species and datasets revealed by blast; the transcripts of P. harmala and close species of the Sapindales order (A), model plants (B), alkaloid plants (C), and SwissProt, NR and Arabidopsis (D) were compared; the number of transcripts with significant hits is presented in each intersection of the Venn diagram; the most common transcripts were observed between the transcripts of P. harmala and the species of Sapindales

Fig. 8

Types of transcriptional elements and regulatory factors of the transcripts of Peganum harmala transcriptome revealed by PlantTFcat; the top types were transcription factor (TF), TF interactor and regulator, chromatin regulator, and chromatin remodeling

Fig. 9

The MAPK signaling pathway of the KEGG database revealed for the transcripts of Peganum harmala transcriptome; shown it was the KEGG map with the highest number of P. harmala transcript mapped against it; the genes are involved in biotic and abiotic stress responses such as stomatal development, ROS (Reactive Oxygen Species) homeostasis, ethylene biosynthesis, defense response, and adaptation; the green boxes show the gene present in the transcriptome of P. harmala

Fig. 10

The plant hormone pathway of the KEGG database revealed for the transcripts of Peganum harmala transcriptome; it was the second pathway with the highest number of P. harmala assigned transcripts; the green boxes show the gene present in the transcriptome of P. harmala