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A new picture of cell wall protein dynamics in elongating cells of Arabidopsis thaliana: confirmed actors and newcomers.

Irshad M, Canut H, Borderies G, Pont-Lezica R, Jamet E - BMC Plant Biol. (2008)

Bottom Line: Apart from expected proteins known to be involved in cell wall extension such as xyloglucan endotransglucosylase-hydrolases, expansins, polygalacturonases, pectin methylesterases and peroxidases, new proteins were identified such as proteases, proteins related to lipid metabolism and proteins of unknown function.This work highlights the CWP dynamics that takes place between the two developmental stages.Finally, putative regulatory mechanisms of protein biological activity are discussed from this global view of cell wall proteins.

View Article: PubMed Central - HTML - PubMed

Affiliation: Surfaces cellulaires et Signalisation chez les Végétaux, UMR 5546 CNRS - UPS-Université de Toulouse, Pôle de Biotechnologie Végétale, 24 chemin de Borde-Rouge, BP 42617 Auzeville, 31326 Castanet-Tolosan, France. muhammad@scsv.ups-tlse.fr

ABSTRACT

Background: Cell elongation in plants requires addition and re-arrangements of cell wall components. Even if some protein families have been shown to play roles in these events, a global picture of proteins present in cell walls of elongating cells is still missing. A proteomic study was performed on etiolated hypocotyls of Arabidopsis used as model of cells undergoing elongation followed by growth arrest within a short time.

Results: Two developmental stages (active growth and after growth arrest) were compared. A new strategy consisting of high performance cation exchange chromatography and mono-dimensional electrophoresis was established for separation of cell wall proteins. This work allowed identification of 137 predicted secreted proteins, among which 51 had not been identified previously. Apart from expected proteins known to be involved in cell wall extension such as xyloglucan endotransglucosylase-hydrolases, expansins, polygalacturonases, pectin methylesterases and peroxidases, new proteins were identified such as proteases, proteins related to lipid metabolism and proteins of unknown function.

Conclusion: This work highlights the CWP dynamics that takes place between the two developmental stages. The presence of proteins known to be related to cell wall extension after growth arrest showed that these proteins may play other roles in cell walls. Finally, putative regulatory mechanisms of protein biological activity are discussed from this global view of cell wall proteins.

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Analysis of proteins extracted by CaCl2 and LiCl from the cell wall fraction prepared from 5 day-old hypocotyls. a. Separation of proteins by cation exchange chromatography. The graph represents amounts of proteins in each fraction eluted by a NaCl gradient (from 0 M to 0.8 M), followed by two steps at 1.2 M and 1.5 M NaCl. Dotted vertical lines show the grouping of chromatography fractions. Ub stands for unfixed fraction, Wa for washes of the column prior to protein elution, numbering to FPLC fractions, and letters (from A to M) to pools analyzed by 1D-E. b. Separation by 1D-E of the total protein extract (total) and of fractions A to M obtained after cation exchange chromatography. Molecular mass markers are on the right. Numbers refer to bands analyzed by MALDI-TOF MS with successful identification (see Additional data file 1).
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Figure 1: Analysis of proteins extracted by CaCl2 and LiCl from the cell wall fraction prepared from 5 day-old hypocotyls. a. Separation of proteins by cation exchange chromatography. The graph represents amounts of proteins in each fraction eluted by a NaCl gradient (from 0 M to 0.8 M), followed by two steps at 1.2 M and 1.5 M NaCl. Dotted vertical lines show the grouping of chromatography fractions. Ub stands for unfixed fraction, Wa for washes of the column prior to protein elution, numbering to FPLC fractions, and letters (from A to M) to pools analyzed by 1D-E. b. Separation by 1D-E of the total protein extract (total) and of fractions A to M obtained after cation exchange chromatography. Molecular mass markers are on the right. Numbers refer to bands analyzed by MALDI-TOF MS with successful identification (see Additional data file 1).

Mentions: Finally, proteins were separated prior to their identification by peptide mass mapping using MALDI-TOF MS and bioinformatics. Since 2D-E is not appropriate for resolving CWPs [12], these proteins were separated using mono-dimensional gel electrophoresis (1D-E). Approximately 60 bands were stained with Coomassie Brilliant Blue (CBB). Fifty two and 67 proteins were respectively identified in the extract from 5 and 11 day-old hypocotyls extracts (Additional data files 1, 2). Because of the limited resolution by 1D-E, many proteins, some of which have a low number of peptides, were identified in each band. In order to improve the separation and identification of proteins, we introduced an additional step prior to 1D-E. Since most CWPs are basic [16], a cation exchange chromatography was performed using an FPLC device (Figures 1A, 2A). Fractions were collected and combined prior to separation by 1D-E (Figures 1B, 2B). At this point, approximately 500 bands were stained with CBB and were further analyzed by MALDI-TOF MS. The proportion of successful protein identification in stained bands was about 70%. Respectively 141 and 109 proteins were identified in 5 and 11 day-old hypocotyls. Many of the proteins were identified in several bands, thus reinforcing their identification. There was a great improvement in the quality of the analysis: (i) the number of identified proteins was doubled; (ii) the quality of the identifications was improved with higher numbers of peptides for identification of most proteins (Additional data files 3 and 4); (iii) the semi-quantification of proteins allowed the comparison of the two samples.


A new picture of cell wall protein dynamics in elongating cells of Arabidopsis thaliana: confirmed actors and newcomers.

Irshad M, Canut H, Borderies G, Pont-Lezica R, Jamet E - BMC Plant Biol. (2008)

Analysis of proteins extracted by CaCl2 and LiCl from the cell wall fraction prepared from 5 day-old hypocotyls. a. Separation of proteins by cation exchange chromatography. The graph represents amounts of proteins in each fraction eluted by a NaCl gradient (from 0 M to 0.8 M), followed by two steps at 1.2 M and 1.5 M NaCl. Dotted vertical lines show the grouping of chromatography fractions. Ub stands for unfixed fraction, Wa for washes of the column prior to protein elution, numbering to FPLC fractions, and letters (from A to M) to pools analyzed by 1D-E. b. Separation by 1D-E of the total protein extract (total) and of fractions A to M obtained after cation exchange chromatography. Molecular mass markers are on the right. Numbers refer to bands analyzed by MALDI-TOF MS with successful identification (see Additional data file 1).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2551616&req=5

Figure 1: Analysis of proteins extracted by CaCl2 and LiCl from the cell wall fraction prepared from 5 day-old hypocotyls. a. Separation of proteins by cation exchange chromatography. The graph represents amounts of proteins in each fraction eluted by a NaCl gradient (from 0 M to 0.8 M), followed by two steps at 1.2 M and 1.5 M NaCl. Dotted vertical lines show the grouping of chromatography fractions. Ub stands for unfixed fraction, Wa for washes of the column prior to protein elution, numbering to FPLC fractions, and letters (from A to M) to pools analyzed by 1D-E. b. Separation by 1D-E of the total protein extract (total) and of fractions A to M obtained after cation exchange chromatography. Molecular mass markers are on the right. Numbers refer to bands analyzed by MALDI-TOF MS with successful identification (see Additional data file 1).
Mentions: Finally, proteins were separated prior to their identification by peptide mass mapping using MALDI-TOF MS and bioinformatics. Since 2D-E is not appropriate for resolving CWPs [12], these proteins were separated using mono-dimensional gel electrophoresis (1D-E). Approximately 60 bands were stained with Coomassie Brilliant Blue (CBB). Fifty two and 67 proteins were respectively identified in the extract from 5 and 11 day-old hypocotyls extracts (Additional data files 1, 2). Because of the limited resolution by 1D-E, many proteins, some of which have a low number of peptides, were identified in each band. In order to improve the separation and identification of proteins, we introduced an additional step prior to 1D-E. Since most CWPs are basic [16], a cation exchange chromatography was performed using an FPLC device (Figures 1A, 2A). Fractions were collected and combined prior to separation by 1D-E (Figures 1B, 2B). At this point, approximately 500 bands were stained with CBB and were further analyzed by MALDI-TOF MS. The proportion of successful protein identification in stained bands was about 70%. Respectively 141 and 109 proteins were identified in 5 and 11 day-old hypocotyls. Many of the proteins were identified in several bands, thus reinforcing their identification. There was a great improvement in the quality of the analysis: (i) the number of identified proteins was doubled; (ii) the quality of the identifications was improved with higher numbers of peptides for identification of most proteins (Additional data files 3 and 4); (iii) the semi-quantification of proteins allowed the comparison of the two samples.

Bottom Line: Apart from expected proteins known to be involved in cell wall extension such as xyloglucan endotransglucosylase-hydrolases, expansins, polygalacturonases, pectin methylesterases and peroxidases, new proteins were identified such as proteases, proteins related to lipid metabolism and proteins of unknown function.This work highlights the CWP dynamics that takes place between the two developmental stages.Finally, putative regulatory mechanisms of protein biological activity are discussed from this global view of cell wall proteins.

View Article: PubMed Central - HTML - PubMed

Affiliation: Surfaces cellulaires et Signalisation chez les Végétaux, UMR 5546 CNRS - UPS-Université de Toulouse, Pôle de Biotechnologie Végétale, 24 chemin de Borde-Rouge, BP 42617 Auzeville, 31326 Castanet-Tolosan, France. muhammad@scsv.ups-tlse.fr

ABSTRACT

Background: Cell elongation in plants requires addition and re-arrangements of cell wall components. Even if some protein families have been shown to play roles in these events, a global picture of proteins present in cell walls of elongating cells is still missing. A proteomic study was performed on etiolated hypocotyls of Arabidopsis used as model of cells undergoing elongation followed by growth arrest within a short time.

Results: Two developmental stages (active growth and after growth arrest) were compared. A new strategy consisting of high performance cation exchange chromatography and mono-dimensional electrophoresis was established for separation of cell wall proteins. This work allowed identification of 137 predicted secreted proteins, among which 51 had not been identified previously. Apart from expected proteins known to be involved in cell wall extension such as xyloglucan endotransglucosylase-hydrolases, expansins, polygalacturonases, pectin methylesterases and peroxidases, new proteins were identified such as proteases, proteins related to lipid metabolism and proteins of unknown function.

Conclusion: This work highlights the CWP dynamics that takes place between the two developmental stages. The presence of proteins known to be related to cell wall extension after growth arrest showed that these proteins may play other roles in cell walls. Finally, putative regulatory mechanisms of protein biological activity are discussed from this global view of cell wall proteins.

Show MeSH
Related in: MedlinePlus