Isolation and comparative proteomic analysis of mitochondria from the pulp of ripening citrus fruit

Xin Li,Zhen Tian,Yingfang Chai,Pengwei Wang,Chunmei Shi,Feng Zhu,Hongbin Yang,Chengyang Li,Rangwei Xu,Xiuxin Deng,Yunjiang Cheng,Yunliu Zeng

doi:10.1038/s41438-021-00470-w

Abstract

Mitochondria are crucial for the production of primary and secondary metabolites, which largely determine the quality of fruit. However, a method for isolating high-quality mitochondria is currently not available in citrus fruit, preventing high-throughput characterization of mitochondrial functions. Here, based on differential and discontinuous Percoll density gradient centrifugation, we devised a universal protocol for isolating mitochondria from the pulp of four major citrus species, including satsuma mandarin, ponkan mandarin, sweet orange, and pummelo. Western blot analysis and microscopy confirmed the high purity and intactness of the isolated mitochondria. By using this protocol coupled with a label-free proteomic approach, a total of 3353 nonredundant proteins were identified. Comparison of the four mitochondrial proteomes revealed that the proteins commonly detected in all proteomes participate in several typical metabolic pathways (such as tricarboxylic acid cycle, pyruvate metabolism, and oxidative phosphorylation) and pathways closely related to fruit quality (such as γ-aminobutyric acid (GABA) shunt, ascorbate metabolism, and biosynthesis of secondary metabolites). In addition, differentially abundant proteins (DAPs) between different types of species were also identified; these were found to be mainly involved in fatty acid and amino acid metabolism and were further confirmed to be localized to the mitochondria by subcellular localization analysis. In summary, the proposed protocol for the isolation of highly pure mitochondria from different citrus fruits may be used to obtain high-coverage mitochondrial proteomes, which can help to establish the association between mitochondrial metabolism and fruit storability or quality characteristics of different species and lay the foundation for discovering novel functions of mitochondria in plants.

Highlights

Mitochondria, whose origin is described by the universally acknowledged endosymbiosis theory[1], play a central role in eukaryotic cells by providing energy in the form of ATP via aerobic respiration[2]
The mitochondrial marker proteins voltage-dependent anion-selective channel protein 1 (VDAC1) and serine hydroxymethyltransferase (SHMT) were readily detected in the samples extracted from the isolated mitochondria; in contrast, the marker proteins predicted to be localized to the cytosol (UGPase), plastids (RbcL), and peroxisomes (Cat) could not be detected (Fig. 2a), indicating that there was no detectable cytosolic, plastidic, or peroxisomal contamination in the isolated mitochondria
The isolated mitochondria exhibited intact membranes and dense matrices without visible contamination by plastids or peroxisomes. These results suggested that the prepared mitochondria were of high purity and could be used for subsequent subproteomic analysis

Summary

Introduction

Mitochondria, whose origin is described by the universally acknowledged endosymbiosis theory[1], play a central role in eukaryotic cells by providing energy in the form of ATP via aerobic respiration[2]. Mitochondria are the major sites for the metabolism of organic acids, amino acids, and lipids as well as the biosynthesis of vitamins and other cofactors, and they are involved in the development and maintenance of fruit quality[3,4]. Multiple metabolic reactions and biochemical processes take place within this relatively small compartment in a well-coordinated way[5]. It has been estimated that mitochondria synthesize less than 5% of the proteins required for their functions; the remaining 95% of required proteins are encoded by the nuclear genome, translated in the cytosol, and imported into the mitochondria.

Results

Discussion

Conclusion