Cell-Specific Expression and Cellular Compartmental Regulation in Camptothecin Biosynthesis

Area of Science:

• Plant molecular biology
• Biochemistry
• Genomics

Background:

• Plant secondary metabolites, like monoterpenoid indole alkaloids (MIAs), are crucial for plant defense and have therapeutic applications.
• Camptothecin (CPT), an anticancer MIA, biosynthesis and regulation via cellular compartmentalization in Ophiorrhiza pumila are not well understood.
• Understanding cell-specific gene expression is key to elucidating complex metabolic pathways.

Purpose of the Study:

• To investigate the cell-specific expression patterns of CPT biosynthetic genes in O. pumila.
• To uncover the regulatory mechanisms of CPT biosynthesis, focusing on cellular compartmentalization.
• To identify novel genes involved in CPT pathway regulation.

Main Methods:

• Single-cell RNA sequencing (scRNA-seq) of 9181 O. pumila leaf cells.
• Marker gene annotation and in situ hybridization for cell classification.
• Gene expression profiling and pseudotime analysis.

Main Results:

• CPT biosynthetic genes are primarily expressed in epidermal and young O. pumila cells.
• Expanded O. pumila STR-TDC gene cluster shows cell-specific specialization for key enzymes strictosidine synthase (STR) and tryptophan decarboxylase (TDC).
• A novel vacuolar transporter gene, OpAVT1, was identified, mediating tryptophan transport and regulating CPT accumulation.

Conclusions:

• Established a cellular-level framework for CPT biosynthesis and regulation in O. pumila.
• OpAVT1 plays a critical role in regulating CPT and other MIA accumulation through vacuolar transport.
• Findings provide insights into the evolution and specialized regulation of plant secondary metabolite pathways.