Isolation of Chromoplasts and Suborganellar Compartments from Tomato and Bell Pepper Fruit

Barsan, Cristina; Kuntz, Marcel; Pech, Jean‐Claude

doi:10.1007/978-1-4939-6533-5_5

Cited by 8 publications

(10 citation statements)

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“…In US alone, tomatoes are a $2.2 billion industry. Tomato is a model system for studying fruit development [118] and recent insights into the dynamics of the tomato's plastid proteome during the differentiation of chloroplasts to chromoplasts have provided important insights in these processes [47,48,51]. In addition, tomato is a model system for the study of the induction of plant defenses associated with wounding, herbivory and pathogen attack [119].…”

Section: Discussionmentioning

confidence: 99%

“…Numerous methods for isolation of intact plastids and sub-fractionation of chloroplast compartments for proteomics studies in Arabidopsis populate our literature today [42,46,84]. In addition, robust methods have been developed for isolation of metabolomics-and proteomics-grade chromoplasts of tomato fruit [47][48][49][50][51]58] (Additional file 1: Table S1). While protocols for isolating chloroplasts for DNA isolation, enzymatic assays and protein import assays have been described [53][54][55][56][57]85], rather surprisingly, few chloroplast large-scale proteomics studies have been reported for tomato leaves [52] (Additional file 1: Table S1).…”

Section: Overview Of the Tomato Chloroplast Isolation Methodsmentioning

confidence: 99%

“…Methods for isolating chloroplasts of proteomics quality from Arabidopsis, pea, spinach, and several monocots have been described [39][40][41][42][43][44][45][46]. In addition, methods for tomato chromoplast isolation have been described and utilized in describing chromoplast transitions during fruit development [47][48][49][50][51]. In contrast, the methods for proteomics-grade tomato chloroplasts are limited [52].…”

Section: Introductionmentioning

confidence: 99%

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Methodology: an optimized, high-yield tomato leaf chloroplast isolation and stroma extraction protocol for proteomics analyses and identification of chloroplast co-localizing proteins

2020

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Background Chloroplasts are critical organelles that perceive and convey metabolic and stress signals to different cellular components, while remaining the seat of photosynthesis and a metabolic factory. The proteomes of intact leaves, chloroplasts, and suborganellar fractions of plastids have been evaluated in the model plant Arabidopsis, however fewer studies have characterized the proteomes of plastids in crops. Tomato (Solanum lycopersicum) is an important world-wide crop and a model system for the study of wounding, herbivory and fruit ripening. While significant advances have been made in understanding proteome and metabolome changes in fruit ripening, far less is known about the tomato chloroplast proteome or its subcompartments. Results With the long-term goal of understanding chloroplast proteome dynamics in response to stress, we describe a high-yielding method to isolate intact tomato chloroplasts and stromal proteins for proteomic studies. The parameters that limit tomato chloroplast yields were identified and revised to increase yields. Compared to published data, our optimized method increased chloroplast yields by 6.7- and 4.3-fold relative to published spinach and Arabidopsis leaf protocols, respectively; furthermore, tomato stromal protein yields were up to 79-fold higher than Arabidopsis stromal proteins yields. We provide immunoblot evidence for the purity of the stromal proteome isolated using our enhanced methods. In addition, we leverage our nanoliquid chromatography tandem mass spectrometry (nanoLC–MS/MS) data to assess the quality of our stromal proteome. Using strict criteria, proteins detected by 1 peptide spectral match, by one peptide, or were sporadically detected were designated as low-level contaminating proteins. A set of 254 proteins that reproducibly co-isolated with the tomato chloroplast stroma were identified. The subcellular localization, frequency of detection, normalized spectral abundance, and functions of the co-isolating proteins are discussed. Conclusions Our optimized method for chloroplast isolation increased the yields of tomato chloroplasts eightfold enabling the proteomics analysis of the chloroplast stromal proteome. The set of 254 proteins that co-isolate with the chloroplast stroma provides opportunities for developing a better understanding of the extensive and dynamic interactions of chloroplasts with other organelles. These co-isolating proteins also have the potential for expanding our knowledge of proteins that are co-localized in multiple subcellular organelles.

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Section: Discussionmentioning

confidence: 99%

Section: Overview Of the Tomato Chloroplast Isolation Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Methodology: an optimized, high-yield tomato leaf chloroplast isolation and stroma extraction protocol for proteomics analyses and identification of chloroplast co-localizing proteins

2020

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“…One of the most commonly used methods is the sucrose gradient. This method is applied on a crude extract of tomato chromoplast which is prepared from around 300 g of tomato fruits subjected to a series of washes, grinding, filtering, and centrifugation steps (Barsan et al 2017). The crude chromoplast suspension is layered on top of the sucrose gradient and subjected to centrifugation allowing the different organelles to be localized in the different fractions (e.g.…”

Section: Plastid Proteomics: From Chloroplast Isolation To Mass Spectmentioning

confidence: 99%

The proteOMIC era: a useful tool to gain deeper insights into plastid physiology

Moreno

2018

Theor. Exp. Plant Physiol.

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Chloroplasts, the green plastid-type present in all photosynthetic organisms, are the physical place where photosynthesis and many other metabolic pathways occur. Chloroplasts are essential for plants, not only by performing photosynthesis but also due to the production of important compounds comprising a great variety of secondary metabolites, lipids, and plant hormones. The production of these compounds is highly regulated and coordinated with development in crops that have fruits with specialized plastids called chromoplasts. Study of plastid biology is essential to understand plant physiology and how plastid biogenesis and development impact plant growth. With the introduction of the genetic and genomic technologies to plant research the discovery and functional characterization of chloroplast proteins was boosted. Nowadays, technologies such as transcriptomics and proteomics are routinely used to assign functions to chloroplast proteins. The generation of high-throughput data sets allows a great increase in our knowledge about many chloroplast processes (e.g. pigment synthesis and accumulation, chloroplast to chromoplast transition, protein degradation) but also the possibility to apply these knowledge to genetically modify plants to improve beneficial traits (e.g. biomass, carotenoid content). The aim of this review is to highlight the importance of proteomic approaches for the study of plastid biology and how this technique speeds up the gain of knowledge in this field.

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“…Isolation of plastids is a prerequisite for the in-depth study of plastid function during citrus fruit ripening. Although there are studies for plastid isolation in limited plant species, including those using sucrose gradient to isolate chromoplasts and suborganellar compartments in different vegetables [ 19 , 20 ] and that using Nycodenz density gradient for plastid isolation in tomato [ 21 ] and sweet orange pulp [ 18 ], isolation of chloroplasts or chromoplasts from the peel of citrus fruit is still very challenging due to the presence of large amounts of essential oils and acidic juices in the fruit. In this work, we identified a citrus variety, the ‘Hua Pi’ kumquat ( Fortunella crassifolia Swingle), whose oil-free property greatly reduces the difficulty of plastid isolation from citrus peel.…”

Section: Introductionmentioning

confidence: 99%

Combining BN-PAGE and microscopy techniques to investigate pigment-protein complexes and plastid transitions in citrus fruit

et al. 2022

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Background Chlorophyll and carotenoids, the most widely distributed lipophilic pigments in plants, contribute to fruit coloration during development and ripening. These pigments are assembled with pigment-protein complexes localized at plastid membrane. Pigment-protein complexes are essential for multiple cellular processes, however, their identity and composition in fruit have yet to be characterized. Results By using BN-PAGE technique in combination with microscopy, we studied pigment-protein complexes and plastid transformation in the purified plastids from the exocarp of citrus fruit. The discontinuous sucrose gradient centrifugation was used to isolate total plastids from kumquat fruit, and the purity of isolated plastids was assessed by microscopy observation and western blot analysis. The isolated plastids at different coloring stages were subjected to pigment autofluorescence observation, western blot, two-dimensional electrophoresis analysis and BN-PAGE assessment. Our results demonstrated that (i) chloroplasts differentiate into chromoplasts during fruit coloring, and this differentiation is accompanied with a decrease in the chlorophyll/carotenoid ratio; (ii) BN-PAGE analysis reveals the profiles of macromolecular protein complexes among different types of plastids in citrus fruit; and (iii) the degradation rate of chlorophyll-protein complexes varies during the transition from chloroplasts to chromoplasts, with the stability generally following the order of LHCII > PS II core > LHC I > PS I core. Conclusions Our optimized methods for both plastid separation and BN-PAGE assessment provide an opportunity for developing a better understanding of pigment-protein complexes and plastid transitions in plant fruit. These attempts also have the potential for expanding our knowledge on the sub-cellular level synchronism of protein changes and pigment metabolism during the transition from chloroplasts to chromoplasts.

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Isolation of Chromoplasts and Suborganellar Compartments from Tomato and Bell Pepper Fruit

Cited by 8 publications

References 17 publications

Methodology: an optimized, high-yield tomato leaf chloroplast isolation and stroma extraction protocol for proteomics analyses and identification of chloroplast co-localizing proteins

Methodology: an optimized, high-yield tomato leaf chloroplast isolation and stroma extraction protocol for proteomics analyses and identification of chloroplast co-localizing proteins

The proteOMIC era: a useful tool to gain deeper insights into plastid physiology

Combining BN-PAGE and microscopy techniques to investigate pigment-protein complexes and plastid transitions in citrus fruit

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