Illuminating the cells:transient transformation of citrus to study gene functions and organelle activities related to fruit quality

Although multiple microscopic techniques have been applied to horticultural research,few studies of individual organelles in living fruit cells have been reported to date.In this paper,we established an efficient system for the transient transformation of citrus fruits using an Agrobacterium-mediated method.Kumquat(Fortunella crassifolia Swingle)was used;it exhibits higher transformation efficiency than all citrus fruits that have been tested and a prolonged-expression window.Fruits were transformed with fluorescent reporters,and confocal microscopy and live-cell imaging were used to study their localization and dynamics.Moreover,various pH sensors targeting different subcellular compartments were expressed,and the local pH environments in cells from different plant tissues were compared.The results indicated that vacuoles are most likely the main organelles that contribute to the low pH of citrus fruits.In summary,our method is effective for studying various membrane trafficking events,protein localization,and cell physiology in fruit and can provide new insight into fruit biology research.

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

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 asγ-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.