Abstract
Tomato is a model for fruit development and ripening. The isolation of intact plastids from this organism is therefore important for metabolic and proteomic analyses. Pepper, a species from the same family, is also of interest since it allows isolation of intact chromoplasts in large amounts. Here, we provide a detailed protocol for the isolation of tomato plastids at three fruit developmental stages, namely, nascent chromoplasts from the mature green stage, chromoplasts from an intermediate stage, and fully differentiated red chromoplasts. The method relies on sucrose density gradient centrifugations. It yields high purity organelles suitable for proteome analyses. Enzymatic and microscopy assays are summarized to assess purity and intactness. A method is also described for subfractionation of pepper chromoplast lipoprotein structures.
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References
Egea I, Barsan C, Bian WP et al (2010) Chromoplast differentiation: current status and perspectives. Plant Cell Physiol 51:1601–1611
Camara B, Hugueney P, Bouvier F et al (1995) Biochemistry and molecular biology of chromoplast development. Int Rev Cytol 163:175–247
Angaman DM, Petrizzo R, Hernandez-Gras F et al (2012) Precursor uptake assays and metabolic analyses in isolated tomato fruit chromoplasts. Plant Methods 8(1):1
Barsan C, Sanchez-Bel P, Rombaldi C et al (2010) Characteristics of the tomato chromoplast revealed by proteomic analysis. J Exp Bot 61:2413–2431
Pateraki I, Renato M, Azcon-Bieto J et al (2013) An ATP synthase harboring an atypical gamma-subunit is involved in ATP synthesis in tomato fruit chromoplasts. Plant J 74:74–85
Wang YQ, Yang Y, Fei ZJ et al (2013) Proteomic analysis of chromoplasts from six crop species reveals insights into chromoplast function and development. J Exp Bot 64:949–961
Zeng YL, Pan ZY, Ding YD et al (2011) A proteomic analysis of the chromoplasts isolated from sweet orange fruits [Citrus sinensis (L.) Osbeck]. J Exp Bot 62:5297–5309
Siddique MA, Grossmann J, Gruissem W et al (2006) Proteome analysis of bell pepper (Capsicum annuum L.) chromoplasts. Plant Cell Physiol 47:1663–1673
Egea I, Bian WP, Barsan C et al (2011) Chloroplast to chromoplast transition in tomato fruit: spectral confocal microscopy analyses of carotenoids and chlorophylls in isolated plastids and time-lapse recording on intact live tissue. Ann Bot 108:291–297
Waters MT, Fray RG, Pyke KA (2004) Stromule formation is dependent upon plastid size, plastid differentiation status and the density of plastids within the cell. Plant J 39:655–667
Barsan C, Zouine M, Maza E et al (2012) Proteomic analysis of chloroplast-to-chromoplast transition in tomato reveals metabolic shifts coupled with disrupted thylakoid biogenesis machinery and elevated energy-production components. Plant Physiol 160:708–725
Bathgate B, Purton ME, Grierson D et al (1985) Plastid changes during the conversion of chloroplasts to chromoplasts in ripening tomatoes. Planta 165:197–204
Schulz A, Knoetzel J, Scheller HV et al (2004) Uptake of a fluorescent dye as a swift and simple indicator of organelle intactness: import-competent chloroplasts from soil-grown Arabidopsis. J Histochem Cytochem 52:701–704
Marti MC, Camejo D, Olmos E et al (2009) Characterisation and changes in the antioxidant system of chloroplasts and chromoplasts isolated from green and mature pepper fruits. Plant Biol 11:613–624
van Wijk KJ, Peltier JB, Giacomelli L (2007) Isolation of chloroplast proteins from Arabidopsis thaliana for proteome analysis. Methods Mol Biol 355:43–48
Hadjeb N, Gounaris I, Price CA (1988) Chromoplast-specific proteins in capsicum-annuum. Plant Physiol 88:42–45
Baerenfaller K, Gruissem W, Baginsky S et al (2008) Chapter 11: Species-dependent proteomics In: von Hargen J (ed) Proteomics sample preparation. Weinheim: Wiley-VCH
Campbell DA, Cockshutt AM, Porankiewicz-Asplund J (2003) Analysing photosynthetic complexes in uncharacterized species or mixed microalgal communities using global antibodies. Physiol Plant 119:322–327
Acknowledgments
The authors are grateful to all participants in the chloroplast-to-chromoplast transition project developed at ENSAT-INRA Toulouse. Paloma Sanchez-Bel, Isabel Egea, Wanping Bian, and Alain Latché have contributed to the isolation of chromoplasts and proteomic analysis. Christian Chervin and Alain Jauneau have carried out the work on confocal laser spectrometry.
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Barsan, C., Kuntz, M., Pech, JC. (2017). Isolation of Chromoplasts and Suborganellar Compartments from Tomato and Bell Pepper Fruit. In: Taylor, N., Millar, A. (eds) Isolation of Plant Organelles and Structures. Methods in Molecular Biology, vol 1511. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6533-5_5
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DOI: https://doi.org/10.1007/978-1-4939-6533-5_5
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Publisher Name: Humana Press, New York, NY
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Online ISBN: 978-1-4939-6533-5
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