A simplified method to isolate rice mitochondria

Yanghong Xu,Jishuai Huang,Fang Yang,Zhiwu Dan,Qiannan Zhang,Jun Hu,Xiaoyi Li,Dinghui Luo,Haijun Xiao,Leilei Peng

doi:10.1186/s13007-020-00690-6

Yanghong Xu, Jishuai Huang + Show 8 more

Open Access

https://doi.org/10.1186/s13007-020-00690-6

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Journal: Plant methods	Publication Date: Nov 4, 2020
Citations: 6	License type: open-access

Affiliation: Wuhan University, Sichuan University

Abstract

BackgroundMitochondria play critical roles in plant growth, development and stress tolerance. Numerous researchers have carried out studies on the plant mitochondrial genome structure, mitochondrial metabolism and nuclear-cytoplasmic interactions. However, classical plant mitochondria extraction methods are time-consuming and consist of a complicated ultracentrifugation procedure with expensive reagents. To develop a more rapid and convenient method for the isolation of plant mitochondria, in this study, we established a simplified method to isolate rice mitochondria efficiently for subsequent studies.ResultsTo isolate rice mitochondria, the cell wall was first disrupted by enzymolysis to obtain the protoplast, which is similar to animal mitochondria. Rice mitochondria were then isolated with a modified method based on the animal mitochondria isolation protocol. The extracted mitochondria were next assessed according to DNA and protein levels to rule out contamination by the nucleus and chloroplasts. Furthermore, we examined the physiological status and characteristics of the isolated mitochondria, including the integrity of mitochondria, the mitochondrial membrane potential, and the activity of inner membrane complexes. Our results demonstrated that the extracted mitochondria remained intact for use in subsequent studies.ConclusionThe combination of plant protoplast isolation and animal mitochondria extraction methods facilitates the extraction of plant mitochondria without ultracentrifugation. Consequently, this improved method is cheap and time-saving with good operability and can be broadly applied in studies on plant mitochondria.

Highlights

Mitochondria play critical roles in plant growth, development and stress tolerance
The results suggested that the treatment overnight at 25 °C could yield more viable protoplasts (Table 1)
The protoplasts were further collected by centrifugation at 600 g for 5 min and resuspended gently in mitochondrial isolation buffer (MIB)

Summary

Introduction

Mitochondria play critical roles in plant growth, development and stress tolerance. Numerous researchers have carried out studies on the plant mitochondrial genome structure, mitochondrial metabolism and nuclearcytoplasmic interactions. A large number of studies have revealed that the reason for mitochondrial deficiency might be the incompatibility between mitochondria and the nucleus or mutations in the mitochondrial genome. These dysfunctions result from disorders of transcriptional, post-transcriptional and translational regulation. Many groups have performed plant mitochondrial omics studies, and the interaction between the nucleus and the mitochondrion has contributed to speciation and cytoplasmic male sterility, which could be applied to explore crop hybrid vigor [15,16,17,18,19,20,21,22]

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