Proteomics Analysis of the Wheat Chloroplast and Sub-Organeller Compartments: Isolation and Fractionation by Using Gradient Centrifugation
Abu Hena Mostafa KamalSwapan Kumar RoySoo-Jeong KwonSang‐Woo KimSeong-Woo ChoKeun-Yook ChungMoon-Soon LeeSun–Hee Woo
0
Citation
0
Reference
12
Related Paper
Keywords:
Isolation
Differential centrifugation
Cell fractionation
Cite
Temperature stress is one of the most common external factors that plants have to adapt to. Accordingly, plants have developed several adaptation mechanisms to deal with temperature stress. Chloroplasts are one of the organelles that are responsible for the sensing of the temperature signal and triggering a response. Here, chloroplasts are purified from low temperature (4° C), control (22° C) and high temperature (30° C) grown Malus x domestica microshoots. The purity of the chloroplast fractions is evaluated by marker proteins, as well as by using in silico subcellular localization predictions. The proteins are digested using filter-aided sample processing and analyzed using nano-LC MS/MS. 733 proteins are observed corresponding to published Malus x domestica gene models and 16 chloroplast genome -encoded proteins in the chloroplast preparates. In ANOVA, 56 proteins are found to be significantly differentially abundant (p < 0.01) between chloroplasts isolated from plants grown in different conditions. The differentially abundant proteins are involved in protein digestion, cytoskeleton structure, cellular redox state and photosynthesis, or have protective functions. Additionally, a putative chloroplastic aquaporin is observed. Data are available via ProteomeXchange with identifier PXD014212.
Malus
Organelle
Cite
Citations (2)
The size of the chloroplast DNA molecule of Arabidopsis thaliana has been determined to be about 153 kb (1). In this chapter, the goal is to provide a step—by—step laboratory procedure for isolating chloroplast DNA from Arabidopsis thaliana with minimal nuclear or mitochondrial DNA contamination. In general, a first step in isolating chloroplast DNA is the homogenization of the plant material followed by a filtration step to remove large-sized cell debris and cell fragments. The filtrate is then centrifuged at low speed to precipitate nuclei and chloroplasts. The intact mitochondria, being smaller in size than chloroplasts, remain in the supernatant. To get rid of nuclear DNA, one of two methods is usually used. In the first, the pellet containing nuclei and chloroplasts is treated with DNase. The latter has access to the nuclear DNA, via nuclear pores of the nuclear membrane, leading to its digestion, but has no access to chloroplast DNA because intact chloroplasts have a nonporous envelope. In the second method, the intact chloroplasts are banded in a sucrose— or a percoll—density—gradient, while the nuclei pellet. Banded chloroplasts are carefully removed. Intact chloroplasts, obtained by either a DNase method or a gradient method, are lysed and their proteins digested with a protease. Digested proteins are removed by organic (phenol/chloroform/isoamyl alcohol) extractions, and the nucleic acids (DNA and RNA) of the chloroplasts precipitated by ethanol.
Nuclear DNA
Cite
Citations (6)
With the completion of the sequencing of the Arabidopsis genome and with the significant increase in the amount of other plant genome and expressed sequence tags (ESTs) data, plant proteomics is rapidly becoming a very active field. We have pursued a high-throughput mass spectrometry-based proteomics approach to identify and characterize membrane proteins localized to the Arabidopsis thaliana chloroplastic envelope membrane. In this study, chloroplasts were prepared from plate- or soil-grown Arabidopsis plants using a novel isolation procedure, and "mixed" envelopes were subsequently isolated using sucrose step gradients. We applied two alternative methodologies, off-line multidimensional protein identification technology (Off-line MUDPIT) and one-dimensional (1D) gel electrophoresis followed by proteolytic digestion and liquid chromatography coupled with tandem mass spectrometry (Gel-C-MS/MS), to identify envelope membrane proteins. This proteomic study enabled us to identify 392 nonredundant proteins. Keywords: chloroplastic envelope membrane • proteome • Arabidopsis thaliana • Gel-C-MS/MS
Bottom-up proteomics
Chloroplast membrane
Cite
Citations (274)
Plant phenotyping to date typically comprises morphological and physiological profiling in a high-throughput manner. A powerful method that allows for subcellular characterization of organelle stoichiometric/functional characteristics is still missing. Organelle abundance and crosstalk in cell dynamics and signaling plays an important role for understanding crop growth and stress adaptations. However, microscopy cannot be considered a high-throughput technology. The aim of the present study was to develop an approach that enables the estimation of organelle functional stoichiometry and to determine differential subcellular dynamics within and across cultivars in a high-throughput manner. A combination of subcellular non-aqueous fractionation and liquid chromatography mass spectrometry was applied to assign membrane-marker proteins to cell compartmental abundances and functions of Pisum sativum leaves. Based on specific subcellular affiliation, proteotypic marker peptides of the chloroplast, mitochondria and vacuole membranes were selected and synthesized as heavy isotope labeled standards. The rapid and unbiased Mass Western approach for accurate stoichiometry and targeted absolute protein quantification allowed for a proportional organelle abundances measure linked to their functional properties. A 3D Confocal Laser Scanning Microscopy approach was developed to evaluate the Mass Western. Two P. sativum cultivars of varying morphology and physiology were compared. The Mass Western assay enabled a cultivar specific discrimination of the chloroplast to mitochondria to vacuole relations.
Organelle
Proteome
Cellular compartment
Cite
Citations (8)
Summary Sample purity is the key for a successful in‐depth analysis of any given subcellular proteome. The suitability of free‐flow electrophoresis to assist conventional, centrifugation‐based techniques in the preparation of plant mitochondria from green and non‐green tissue was assessed by various means, including functional assays, immunoblots, electron microscopy and differential gel electrophoresis. Results indicated a significant increase in purity of the mitochondrial samples, highlighted specific contaminants previously reported as mitochondrial proteins, and also pointed to new means for separating plastids and peroxisomes from mitochondria in plant organellar extracts by exploiting differences in surface charge. This approach has the potential to allow a deeper and more comprehensive investigation of the Arabidopsis organellar proteomes, by providing a second dimension of separation based on surface charge in addition to conventional centrifugation purification protocols relying on size and density.
Proteome
Free-flow electrophoresis
Differential centrifugation
Organelle
Cite
Citations (107)
We established and elaborated on a method to enrich the membrane proteome of mature pollen from economically relevant crop using the example of Solanum lycopersicum (tomato). To isolate the pollen protein fraction enriched in membrane proteins, a high salt concentration (750 mM of sodium chloride) was used. The membrane protein-enriched fraction was then subjected to shotgun proteomics for identification of proteins, followed by in silico analysis to annotate and classify the detected proteins.
Shotgun proteomics
Proteome
Shotgun
Cite
Citations (3)
A systematic strategy was developed for the proteomic analysis of wheat chloroplast protein complexes. First, comprehensive centrifugation methods were utilized for the exhaustive isolation of thylakoid, envelope, and stromal fractions. Second, 1% n-dodecyl-β-D-maltoside was selected from a series of detergents as the optimal detergent to dissolve protein complexes effectively from membranes. Then, blue native polyacrylamide gel electrophoresis (BN-PAGE) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were improved to separate and analyze the protein complexes. By this systematic strategy, envelopes, thylakoids, and stromata were enriched effectively from chloroplasts in the same process, and more than 18 complexes were obtained simultaneously by BN-PAGE. Finally, thylakoid protein complexes were further analyzed by BN/SDS-PAGE, and nine complex bands and 40 protein spots were observed on BN-PAGE and SDS-PAGE respectively. Our results indicate that this new strategy can be used efficiently to analyze the proteome of chloroplast protein complexes and can be applied conveniently to the analysis of other subcellular protein complexes.
Proteome
Sodium dodecyl sulfate
Cite
Citations (8)
Cite
Citations (0)
Cite
Citations (24)