Plant proteome analysis

Cánovas, Francisco M.; Dumas‐Gaudot, Eliane; Recorbet, Ghislaine; Jorrı́n, Jesús; Mock, Hans‐Peter; Rossignol, Michel

doi:10.1002/pmic.200300602

Cited by 262 publications

(146 citation statements)

References 172 publications

(121 reference statements)

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“…The increasing impact of proteomic studies on plant biology 15,16 has triggered renewed interest in organelle purification and led to two independent proteomic studies focused on intact vacuoles derived from different plant tissues 17,18 . In contrast to a relatively intricate protocol describing intact vacuole purification from an Arabidopsis cell suspension 18 , we present a simple, reliable method for vacuole isolation from Arabidopsis leaf mesophyl protoplasts in great detail 17 .…”

Section: Introductionmentioning

confidence: 99%

Isolation of intact vacuoles from Arabidopsis rosette leaf–derived protoplasts

et al. 2007

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Vacuoles are very prominent compartments within plant cells, and understanding of their function relies on knowledge of their content. Here, we present a simple vacuole purification protocol that was successfully used for large-scale isolation of vacuoles, free of significant contamination from other endomembrane compartments. This method is based on osmotic and thermal disruption of mesophyl-derived Arabidopsis protoplasts, followed by a density gradient fractionation of the cellular content. The whole procedure, including protoplast isolation, takes approximately 6 h. INTRODUCTIONLarge-scale isolation and characterization of cellular organelles offers excellent opportunities in plant systems biology 1 . Detailed qualitative and quantitative knowledge of the composition of a particular organelle allows for investigation of subcellular pathways through the identification of novel protein components and the analysis of cellular sinks. This also facilitates detailed comparative studies between species or between mutant and wild-type plants. In contrast to some organelles, such as mitochondria and chloroplasts, that are relatively easy to obtain in a pure form 2,3 , plant vacuoles are extremely fragile, and their isolation using conventional tissue homogenization and fractionation schemes can be remarkably challenging and often results in a subcellular fraction without the same properties as intact mature vacuoles 4 . The first successful technique to overcome these difficulties was based on protoplast isolation and subsequent gentle release of vacuoles by osmotic shock 5,6 . Later large-scale isolation protocols employed gradient fractionation of osmotically released organelles 7,8 , and the method was further optimized by application of both osmotic and thermal shocks to the isolated protoplasts 9,10 . As an alternative to the protoplast methods, which are applicable only to relatively soft tissues, vacuoles can be isolated by slicing the plant material and releasing contents into a medium with a perfectly adjusted osmoticum 11 . In subsequent studies, numerous vacuolar constituents have been determined, including sugars and amino acids 12 , hydrolases 13 and ions 14 .

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Section: Introductionmentioning

confidence: 99%

Isolation of intact vacuoles from Arabidopsis rosette leaf–derived protoplasts

et al. 2007

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“…Nonextractable proteins are membrane associated or integrated basic proteins with a high sugar content [22] and nonextractable storage proteins. For a more complete extraction of the plant proteome, special procedures must be used [23].…”

Section: The Inxuence Of Fusarium Infection On the Quantitative Protementioning

confidence: 99%

The influence of Fusarium infection and growing location on the quantitative protein composition of (part I) emmer (Triticum dicoccum)

Eggert

Wieser²,

Pawelzik

2010

Eur Food Res Technol

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The eVect of the fungal infection by Fusarium graminearum and Fusarium culmorum of emmer and wheat cultivars on their total protein content and the protein fractions albumins + globulins, gliadins and glutenins and their diVerent protein types was investigated. In addition, the inXuence of two diVerent locations on the quantitative protein composition was evaluated. The results showed that Fusarium infection changed the content of gliadins and glutenins in emmer and wheat. The wheat glutenin fractions and types were found to be more strongly aVected by the Fusarium spp. infection than the glutenin fractions and types in emmer cultivars in spite of the wheat's lower degree of infection. The nitrogen supply at the two locations was associated with an increase in the gliadin content in emmer and an increase in the glutenin content in emmer and wheat. Nitrogen availability, a factor which promotes gene expression, resulted here in a species-speciWc eVect on the gliadin/glutenin ratio.

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“…1,2 Numerous proteomic analyses have been undertaken in Arabidopsis whole plants and cell suspension cultures subjected to a variety of stimuli and physiological conditions, as well as extensive profiling of the protein composition of purified subcellular organelles. [3][4][5][6][7][8] To increase proteomic coverage, all of these studies invested significant efforts to enrich and fractionate plant proteins obtained from whole cells or purified plastids, mitochondria, and membrane components prior to MS-based proteomic analysis.…”

Section: Introductionmentioning

confidence: 99%

Membrane Proteomic Analysis of Arabidopsis thaliana Using Alternative Solubilization Techniques

et al. 2007

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This study presents a comparative proteomic analysis of the membrane subproteome of whole Arabidopsis seedlings using 2% Brij-58 or 60% methanol to enrich and solubilize membrane proteins for strong cation exchange fractionation and reversed-phase liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of 441 proteins were identified by our Brij-58 method, and 300 proteins were detected by our methanol-based solubilization approach. Although the total number of proteins obtained using the nonionic detergent was higher than the total obtained by organic solvent, the ratio of predicted membrane proteins to total proteins identified indicates up to an 18.6% greater enrichment efficiency using methanol. Using two different bioinformatics approaches, between 31.0 and 40.0% of the total proteins identified by the methanol-based method were classified as containing at least one putative transmembrane domain as compared to 22.0-23.4% for Brij-58. In terms of protein hydrophobicity as determined by the GRAVY index, it was revealed that methanol was more effective than Brij-58 for solubilizing membrane proteins ranging from -0.4 (hydrophilic) to +0.4 (hydrophobic). Methanol was also approximately 3-fold more effective than Brij-58 in identifying leucine-rich repeat receptor-like kinases. The ability of methanol to effectively solubilize and denature both hydrophobic and hydrophilic proteins was demonstrated using bacteriorhodopsin and cytochrome c, respectively, where both proteins were identified with at least 82% sequence coverage from a single reversed-phase LC-MS/MS analysis. Overall, our data show that methanol is a better alternative for identifying a wider range of membrane proteins than the nonionic detergent Brij-58.

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Plant proteome analysis

Cited by 262 publications

References 172 publications

Isolation of intact vacuoles from Arabidopsis rosette leaf–derived protoplasts

Isolation of intact vacuoles from Arabidopsis rosette leaf–derived protoplasts

The influence of Fusarium infection and growing location on the quantitative protein composition of (part I) emmer (Triticum dicoccum)

Membrane Proteomic Analysis of Arabidopsis thaliana Using Alternative Solubilization Techniques

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