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Biochemistry of Diplonemids.

Matus Valach¹, Gertraud Burger¹, Drahomíra Faktorová^2,3

¹Department of Biochemistry and Robert-Cedergren Centre for Bioinformatics and Genomics, Université de Montréal, Montréal, QC, Canada.

Methods in Molecular Biology (Clifton, N.J.)

|February 2, 2026

Summary

Researchers developed new protocols for studying diplonemids, marine flagellates with unique mitochondrial genomes. These methods enable detailed analysis of their unusual mitochondrial gene expression and metabolic functions.

Keywords:

Bioenergetics Diplonema Diplonemid Mitochondria Mitoribosome Nanopore sequencing Reactive oxygen species Respiration Stable-isotope labeling Subcellular fractionation

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Area of Science:

Protistology
Molecular Biology
Biochemistry

Background:

Diplonemids are marine flagellates known for their unusual mitochondrial genomes and gene expression.
Understanding these unique mitochondrial processes is crucial but challenging.
Previous experimental procedures required optimization for studying these organisms.

Purpose of the Study:

To establish and optimize a comprehensive set of experimental protocols for diplonemids.
To facilitate the study of mitochondrial genome and gene expression in diplonemids.
To enable detailed investigation of metabolic functions in Paradiplonema papillatum.

Main Methods:

DNA preparation for long-read sequencing of mitochondrial and nuclear DNA.
Isolation of mitochondria and separation of mitochondrial ribosomes.
Assays for metabolic functions: respiration, membrane potential, peroxide-scavenging.
Stable-isotope labeling for tracking metabolic routes.

Main Results:

A robust collection of protocols for analyzing diplonemid mitochondrial biology is now available.
These protocols cover DNA sequencing, organelle isolation, and functional metabolic assays.
The methods allow for in-depth study of metabolic pathways and mitochondrial activities.

Conclusions:

The presented protocols provide essential tools for advancing research on diplonemid mitochondrial biology.
This work enhances our ability to investigate the unique molecular and metabolic characteristics of diplonemids.
Future studies can now more effectively explore the eccentric mitochondrial processes in this group.