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AT_Chloro: The first AMT database of chloroplast proteins

​Researchers at the Cell & Plant Physiology Laboratory and at the EDyP team developed an innovative proteomics strategy targeting various highly-purified chloroplast compartments. They then went on to develop AT_Chloro, the first AMT database dedicated to the model plant Arabidopsis thaliana.​​

Published on 6 March 2010
Most renewable carbon is fixed by photosynthetic organisms through their chloroplasts. Plastids are semi-autonomous organelles found in terrestrial plants, algae, and some protists. Being the place where photosynthesis occurs, chloroplasts perform many other functions such as the synthesis of fatty acids, lipids, amino acids, vitamins, etc... These biosyntheses require the coordinated action of three compartments: the thylakoids, the stroma and the envelope. Deepening knowledge of the chloroplast metabolic pathways and their regulation is considered as essential in the prospect of a better use of plant biomass. Finally, understanding the metabolic processes of the chloroplast is essential if one wants to analyze the impact of environmental change on plants. In this context, proteomic analysis has proven to be the tool of choice to advance significantly the understanding of chloroplast.

For several years, researchers in Plant Cell & Physiology laboratory and Étude de la Dynamique des Protéomes team contribute significantly to the establishment of directories for various protein cell compartments of Arabidopsis thaliana. However, one of the current challenges in proteomics is the comparative studies to monitor dynamic processes. Among the strategies of quantitative proteomic analyzes that have been developed, the AMT (Accurate Mass Tags and time) method (see the thematic letter on the Analyse protéomique) and the so-called Spectral count method allowed these researchers to establish the first AMT database for a specific organelle of the plant cell, the chloroplast.

The AT_Chloro database was established achieving nearly 500 analyzes of highly purified fractions of the three major subcompartments of the chloroplast (envelope, stroma and thylakoids). Specific information have been obtained for 10,654 distinct peptides (
Figure 1) corresponding to 1,323 validated proteins. These data were then structured in AT_Chloro, a database specific of the chloroplast from Arabidopsis thaliana. The creation of this database will allow performing further analyses by nanoLC-FT-MS, avoiding the systematic use of MS / MS, thus significantly increasing the speed of analysis and coverage of the studied proteome. From the perspective of comparative proteomic analyzes, the AMT method has the advantage to directly compare the intensities of MS signals obtained when analyzing different and complex protein samples.

Figure 1: Peptide mass map of ions (peptides) detected by LC-MS in the chloroplast.

X-axis: retention time of the peptides on the nanoLC column (min). Y-axis: isotopic mass of the different peptides (Da). Different charge states (z) of the peptides are represented by different colours: z = 1 in blue, z = 2 in red, z = 3 in green.

The combination of AMT and Spectral Count allowed obtaining, from a single analysis, data identification and quantification. This strategy implemented for the first time has allowed identifying the of subplastidial localization of identified proteins to build the AT_Chloro dabase.
In this study, a main effort was specifically made for the annotation of proteins from the chloroplast envelope, the site of interaction between plastids and the rest of the cell. Thus, nearly 500 proteins (about 1323 proteins identified in the chloroplast) were identified in the envelope while they correspond to (in mass) only 1-2% of the chloroplast proteins. These results confirm the functional importance of this membrane system. The present proteomic analysis has allowed putting the identified proteins in the functional context of metabolic pathways, and of the role that envelope, stroma or thylakoids play in the chloroplast function (
Figure 2).

Figure 2: : Subcellular metabolism of terpene compounds in the chloroplast deduced from the semi-quantitative proteomic data.
Proteomic analysis using "spectral count" shows that the first steps (methylerythritol phosphate pathways, the shikimate or protoporphyrinogen IX, for example) take place in the stroma (the proteins are indicated by orange circles) and then, as soon as molecules become hydrophobic, metabolic pathways involve membrane proteins: either from the thylakoids (green circles) or the envelope (yellow circles). The blue circles correspond to proteins whose localization was not possible. These results exemplify the role of the envelope in the biosynthesis of carotenoids (precursors among others of the ABA hormone), of prenylquinones (plastoquinone, alpha-tocopherol) and of protochlorophyllide, a precursor of the chlorophyll.

The AT_Chloro database goes beyond a simple repertoire of proteins by introducing semi-quantitative data which allow determining in which compartment (envelope, stroma thylakoids) is localized a chloroplast protein. It is therefore possible to specify the role of each compartment in the chloroplast metabolic pathways.
• The AT_Chloro database, which contains a large amount of information on the proteins of the chloroplast envelope, is a unique knowledge database on this membrane system.
• The AT_Chloro database represents a significant step in paving the way for systematic quantitative studies aiming to compare the proteomes of chloroplast mutants or to follow the dynamics of the proteome during the plant development or in plants subjected to changes in their environment.

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