Journal Article

A Proteomic Approach to Apoplastic Proteins Involved in Cell Wall Regeneration in Protoplasts of <i>Arabidopsis</i> Suspension-cultured Cells

Hye-Kyoung Kwon, Ryusuke Yokoyama and Kazuhiko Nishitani

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 46, issue 6, pages 843-857
Published in print June 2005 | ISSN: 0032-0781
Published online June 2005 | e-ISSN: 1471-9053 | DOI: http://dx.doi.org/10.1093/pcp/pci089
A Proteomic Approach to Apoplastic Proteins Involved in Cell Wall Regeneration in Protoplasts of Arabidopsis Suspension-cultured Cells

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To clarify the mechanisms of cell wall construction, we used a proteomic approach to investigate the proteins secreted into cell wall spaces during cell wall regeneration from the protoplasts of Arabidopsis suspension-cultured cells. We focused on cell wall proteins loosely bound to the cell wall architecture and extractable with 1 M KCl solutions from: (i) native suspension cultured cells; (ii) protoplasts that had been allowed to regenerate their cell walls for 1 h; and (iii) protoplasts allowed to regenerate their cell walls for 3 h. We adopted a non-destructive extraction procedure without disrupting cellular integrity, thereby avoiding contamination from cytoplasmic proteins. Using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and matrix-assisted laser desorption ionization-time-of-flight/mass spectrometry (MALDI-TOF/MS), we separated, mapped and identified 71 proteins derived from the native cell wall, and 175 and 212 proteins derived from the 1 and 3 h regenerated protoplasts, respectively. Quite different sets of proteins with differing status of their post-translational modifications, including phosphorylation and glycosylation, were identified in the three protein fractions. This indicated dynamic in muro changes in the cell wall proteins during cell wall regeneration in the protoplasts. The analysis revealed a set of enzymes specifically involved in cell wall expansion and construction in suspension-cultured cells. This approach has also determined a set of cell wall proteins that had not been predicted to be localized in cell wall spaces.

Keywords: Arabidopsis; Cell wall regeneration; Extracellular proteins; Post-translational processing; Proteomics; Suspension cultured cells; CBB, Coomassie brilliant blue; 2-D PAGE, two-dimensional polyacrylamide gel electrophoresis; DTT, dithiothreitol; FDA, fluorescein diacetate; G6PDH, glucose-6-phosphate dehydrogenase; IEF, isoelectric focusing; MALDI-TOF/MS, matrix-assisted laser desorption ionization-time-of-flight/mass spectrometry; SEM, scanning electron microscope; xyloglucan endotransglucosylase/hydrolase, XTH.

Journal Article.  10073 words.  Illustrated.

Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry

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