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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 ?-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.

SUBMITTER: Li X

PROVIDER: S-EPMC7848011 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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

Li Xin X Chai Yingfang Y Yang Hongbin H Tian Zhen Z Li Chengyang C Xu Rangwei R Shi Chunmei C Zhu Feng F Zeng Yunliu Y Deng Xiuxin X Wang Pengwei P Cheng Yunjiang Y

Horticulture research 20210201 1

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, includin ...[more]

PMID: 33518707

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Project description:BackgroundInterspecific hybridizations and admixtures were key in Citrus domestication, but very little is known about their impact at the transcriptomic level. To determine the effects of genome introgressions on gene expression, the transcriptomes of the pulp and flavedo of three pure species (citron, pure mandarin and pummelo) and four derived domesticated genetic admixtures (sour orange, sweet orange, lemon and domesticated mandarin) have been analyzed at color break.ResultsMany genes involved in relevant physiological processes for domestication, such sugar/acid metabolism and carotenoid/flavonoid synthesis, were differentially expressed among samples. In the low-sugar, highly acidic species lemon and citron, many genes involved in sugar metabolism, the TCA cycle and GABA shunt displayed a reduced expression, while the P-type ATPase CitPH5 and most subunits of the vacuolar ATPase were overexpressed. The red-colored species and admixtures were generally characterized by the overexpression in the flavedo of specific pivotal genes involved in the carotenoid biosynthesis, including phytoene synthase, ζ-carotene desaturase, β-lycopene cyclase and CCD4b, a carotenoid cleavage dioxygenase. The expression patterns of many genes involved in flavonoid modifications, especially the flavonoid and phenylpropanoid O-methyltransferases showed extreme diversity. However, the most noticeable differential expression was shown by a chalcone synthase gene, which catalyzes a key step in the biosynthesis of flavonoids. This chalcone synthase was exclusively expressed in mandarins and their admixed species, which only expressed the mandarin allele. In addition, comparisons between wild and domesticated mandarins revealed that the major differences between their transcriptomes concentrate in the admixed regions.ConclusionIn this work we present a first study providing broad evidence that the genome introgressions that took place during citrus domestication largely shaped gene expression in their fruits.

Dataset Information

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

Publications

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

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