De Novo Transcriptomic Profiling of Piper chaba: Elucidating the Genetic Basis of Piperine Biosynthesis and Metabolic...

Medicinal plants serve as invaluable sources of bioactive compounds, yet the molecular basis of their secondary metabolite biosynthesis remains largely unexplored. Piper chaba Hunter, an important but understudied member of the Piperaceae family, is known for its pharmacologically active alkaloids, particularly piperine. To our knowledge, this is the first de novo transcriptomic profiling of spike, leaf, and root tissues of P. chaba to uncover the genetic pathways regulating its metabolite production. Piperine, a major bioactive compound, was quantified using UPLC. The highest concentration was observed in the spike (331.3 mg/g), followed by the root (10.3 mg/g) and leaves (2.82 mg/g). High-quality RNA sequencing of leaves, roots, and spikes using next-generation sequencing (NGS) generated 228,481 transcripts, and 184,574 unigenes were identified after redundancy removal. Coding sequences (CDSs) derived from these unigenes were annotated using BLASTX and KEGG databases, which highlighted significant metabolic pathways, including those related to piperine biosynthesis. Thirteen candidate genes potentially associated with the piperine biosynthetic pathway were identified based on transcriptome annotation and pathway analysis. Validation of nine selected genes, including farnesyl pyrophosphate synthase and piperic acid synthase, was performed through qRT-PCR using the 2^-ΔΔCt method, supporting their expression patterns potentially associated with piperine and related metabolite biosynthesis. Functional annotation categorized the CDS into Gene Ontology domains, with transcription factors such as bHLH and NAC families playing prominent roles in metabolic regulation. Additionally, 5050 SSRs were identified, offering potential markers for genetic studies. This pioneering study establishes a molecular framework for understanding the biosynthetic pathways of P. chaba, providing valuable insights for its application in sustainable medicine and agriculture.