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

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.

Fig. 1. Workflow diagram for the isolation and purification of mitochondria from the pulp of different citrus species.

a–d Anatomic structures of four citrus species used for mitochondrial preparation: (a) satsuma mandarin, (b) ponkan mandarin, (c) sweet orange, and (d) shatian pummelo. Bars = 10 mm. e Differential and Percoll density gradient centrifugation procedures for preparing highly pure mitochondria from the pulp of different citrus species. Tissue homogenization, formulation of extraction buffer, and centrifugation steps were optimized to obtain high-quality mitochondria

Fig. 2. Purity evaluation of isolated mitochondria.

a Western blot analysis of mitochondrial enriched fractions (mito) and total pulp protein extracts (pulp) of different citrus species. A total of 10 μg of mitochondrial proteins and pulp proteins were separated by 10% SDS-PAGE and blotted onto PVDF membranes. Blots were performed using different marker antibodies against VDAC1 (mitochondrial outer membrane), SHMT (mitochondrial matrix), UGPase (cytoplasm), RbcL (plastid), and Cat (peroxisome). Uncropped blot images are shown in Supplementary Fig. S6. b–e Transmission electron microscopy (TEM) images of isolated mitochondria from (b) satsuma mandarin, (c) ponkan mandarin, (d) sweet orange, and (e) shatian pummelo. Bars = 1 μm

Fig. 3. Characterization and functional classification of four citrus mitochondrial proteomes.

a Venn diagram of the overlapping proteins identified from at least two biological replicates in each species. A total of 1614 proteins were commonly identified in four species. Detailed information on 3353 nonredundant proteins identified from the four species is given in Supplementary Table S2. b KEGG analysis of the 1614 overlapping proteins indicated in (a). Pathways related to energy metabolism, such as oxidative phosphorylation, TCA cycle, and pyruvate metabolism, were significantly enriched. c Functional distribution of the 3353 proteins identified in this study based on the functional classification of Heazlewood et al.. These proteins can be divided into 12 major functional groups and mainly participate in metabolism (29.0%), protein fate (15.7%), protein synthesis (9.4%), energy (7.8%), and cellular transport and transport mechanisms (7.4%)

Fig. 4. Principal component analysis and hierarchical clustering analysis of differentially abundant proteins among four citrus mitochondrial proteomes.

a A total of 2716 DAPs were screened among four species using the fold change method (> 2 or < 0.5) and P value < 0.05 with Student’s t-test. Similar protein expression profiles were observed among three biological replicates for each species. b The protein expression patterns showed a relatively high degree of similarity between satsuma and ponkan, as well as between orange and pummelo. Detailed information on the 2716 DAPs between species is listed in Supplementary Table S3

Fig. 5. Comparison of mitochondrial proteomes between loose-skinned and tight-skinned citrus fruits.

a Venn diagram of the overlapping DAPs in different combinations of species. There were 584 DAPs commonly detected in all four combinations. After the exclusion of the DAPs between species within the same category, a total of 522 proteins were screened as DAPs between loose-skin and tight-skin citrus fruits. b Number of DAPs between loose-skinned and tight-skinned citrus fruits. Detailed information on the 522 DAPs between loose-skinned and tight-skinned citrus fruits is listed in Supplementary Table S5. c KEGG analysis of DAPs between loose-skinned and tight-skinned citrus fruits. Pathways associated with fatty acid and amino acid metabolism were significantly enriched

Fig. 6. Subcellular localization study of ten selected proteins identified in this study.

a–j Ten proteins identified in this study (Supplementary Table S6) were fused in frame with the GFP fluorescence tag and then transiently coexpressed with the mitochondrial marker in tobacco leaves. The left lane shows the GFP fluorescence signal, the center lane shows the mCherry fluorescence of the mitochondrial marker, and the right lane shows an overlay between the two types of fluorescence. Among them, Cs1g19460.1 (b) and Cs2g03620.1 (c) were likely to be partially mitochondria-localized or peripherally associated with mitochondria

Fig. 7. Major similarities and differences in mitochondrial metabolism between loose-skinned and tight-skinned citrus fruits.

Proteins identified in all species were mainly involved in oxidative phosphorylation, the TCA cycle and pyruvate metabolism, indicating that mitochondrial energy metabolism is conserved among different citrus fruits. The DAPs between loose-skinned and tight-skinned citrus fruits were mainly associated with fatty acid and amino acid metabolism, implying differences in mitochondrial metabolism between the two types of citrus fruit