De novo transcriptome profiling revealing genes involved in piperine biosynthetic pathway in Piper longum L

Abstract

Piper longum, commonly known as long pepper, is highly valued for its bioactive alkaloid piperine, which has diverse pharmaceutical and culinary applications. In this study, we used high-throughput sequencing and de novo transcriptome assembly to analyze the transcriptomes of P. longum leaves, roots, and spikes. Our dataset consisted of 173,381 high-quality transcripts, with functional annotations highlighting key pathways involved in lysine biosynthesis and secondary metabolite production. We identified 8041 simple sequence repeats (SSRs), primarily trinucleotide repeats, adding valuable genetic markers. Additionally, we uncovered 21,235 transcription factors (TFs) across 65 families, including bHLH, NAC, and MYB, which are essential in regulating metabolism. KEGG pathway analysis categorized 4730 transcripts into 377 pathways, focusing on piperine biosynthesis, including phenylpropanoid and L-lysine metabolism. Our research identified 14 expressed genes encoding enzymes crucial for tropane, piperidine, and pyridine alkaloid biosynthesis, shedding light on piperine production mechanisms. High-performance liquid chromatography (HPLC) revealed varying piperine concentrations, with spikes showing the highest content, followed by roots and leaves. This comprehensive analysis of the P. longum transcriptome offers valuable insights into the genetics and regulatory networks underlying piperine biosynthesis, identifying molecular markers and genes that hold significant potential for enhancing the plant's biological and pharmaceutical applications.

Keywords: Piper longum; Biosynthetic pathways; Illumina; Piperine; Secondary metabolites; Transcriptome analysis.

Fig. 1

Graphical representation of analysis of piperine content in different parts of P. longum using HPLC. The three replicates ± SE is shown by the error bars above the means.

Fig. 2

Length distribution of P. longum transcripts. Y axis represents the length range whereas the X axis represents the number of transcripts.

Fig. 3

Homologous Resemblance in P. longum: Evaluation of unigene similarity based on annotations using the Nr database. The figure displays the distribution of E-values for the best BLAST hits associated with each unigene (E-value < 1e-5), the distribution of similarities for the top BLAST hits per unigene, and the species distribution of the most closely related sequence results for each unigene.

Fig. 4

Comparative Gene Ontology (GO) classifications of CDS with functional annotations commonly expressed across various tissues in the P. longum transcriptome. These genes were categorized into three principal domains: cellular component (CC), molecular function (MF), and biological process (BP).

Fig. 5

KEGG functional pathway of Piper longum. The Y axis represents the number of CDS whereas the X axis represents the KEGG functional pathway categories.

Fig. 6

Classification based on categories of secondary metabolite biosynthesis. The Y axis represents the different biosynthetic pathways whereas the X axis represents the number of CDS.

Fig. 7

The pictorial representation of the proposed piperine biosynthesis pathway based on prior studies, incorporating the CDS found in P. longum.

Fig. 8

Distribution of different transcription factors in CDS of Piper longum. Y axis represents the transcription factor families and X axis represents the number of transcription factors.

Fig. 9

Validation of selected genes involved in piperine biosynthesis in P. longum (spike) through qRT-PCR. The Y-axis depicts the log2 fold change of selected differentially expressed genes and the X-axis depicts the genes from the transcriptome.