Multiplex staining of 2-DE gels for an initial phosphoproteome analysis of germinating seeds and early grown seedlings from a non-orthodox spp.: Quercus ilex L. subsp. ballota [Desf.] Samp.
As a preliminary step in the phosphoproteome analysis of germinating seeds (0, and 24 h after seed imbibition) and early grown seedlings (216 h after seed imbibition) from a non-orthodox spp. Quercus ilex, a multiplex (SYPRO-Ruby and Pro-Q DPS) staining of high-resolution 2-DE gels was used. By usin...
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doaj-52115c09bf304570a797334ee07b0ef12020-11-24T22:29:40ZengFrontiers Media S.A.Frontiers in Plant Science1664-462X2015-08-01610.3389/fpls.2015.00620146852Multiplex staining of 2-DE gels for an initial phosphoproteome analysis of germinating seeds and early grown seedlings from a non-orthodox spp.: Quercus ilex L. subsp. ballota [Desf.] Samp.María Cristina Romero-Rodriguez0María Cristina Romero-Rodriguez1Nieves eAbril2Rosa eSánchez-Lucas3Jesus V eJorrin Novo4University of CordobaUniversidad Nacional de AsuncionBiochemistry and Molecular BiologyUniversity of CordobaUniversity of CordobaAs a preliminary step in the phosphoproteome analysis of germinating seeds (0, and 24 h after seed imbibition) and early grown seedlings (216 h after seed imbibition) from a non-orthodox spp. Quercus ilex, a multiplex (SYPRO-Ruby and Pro-Q DPS) staining of high-resolution 2-DE gels was used. By using this protocol it was possible to detect changes in protein-abundance and/or phosphorylation status. This simple approach could be a good complementary alternative to the enrichment protocols used in the search for phosphoprotein candidates. While 482 spots were visualized with SYPRO-Ruby, 222 were with Pro-Q DPS. Statistically significant differences in spot intensity were observed among samples, these corresponding to 85 SYPRO-Ruby-, 20 Pro-Q-DPS-, and 35 SYPRO-Ruby and Pro-Q-DPS-stained spots. Fifty-five phosphoprotein candidates showing qualitative or quantitative differences between samples were subjected to MALDI-TOF-TOF MS analysis, with 20 of them being identified. Identified proteins belonged to five different functional categories, namely: carbohydrate and amino acid metabolism, defence, protein folding and oxidation-reduction processes. With the exception of a putative cyclase, the other nineteen proteins had at least one orthologous phosphoprotein in A. thaliana, M. truncatula, N. tabacum and G. max. Out of the twenty identified, seven showed differences in intensity in Pro-Q-DPS but not in SYPRO-Ruby-stained gels, including enzymes of the glycolysis and amino acid metabolism. This bears out that theory the regulation of these enzymes occurs at the post-translational level by phosphorylation with no changes at the transcriptional or translational level. This is different from the mechanism reported in orthodox seeds, in which concomitant changes in abundance and phosphorylation status have been observed for these enzymes.http://journal.frontiersin.org/Journal/10.3389/fpls.2015.00620/fullGerminationphosphoproteomicspost translational modificationrecalcitrant seedsHolm oak |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
María Cristina Romero-Rodriguez María Cristina Romero-Rodriguez Nieves eAbril Rosa eSánchez-Lucas Jesus V eJorrin Novo |
spellingShingle |
María Cristina Romero-Rodriguez María Cristina Romero-Rodriguez Nieves eAbril Rosa eSánchez-Lucas Jesus V eJorrin Novo Multiplex staining of 2-DE gels for an initial phosphoproteome analysis of germinating seeds and early grown seedlings from a non-orthodox spp.: Quercus ilex L. subsp. ballota [Desf.] Samp. Frontiers in Plant Science Germination phosphoproteomics post translational modification recalcitrant seeds Holm oak |
author_facet |
María Cristina Romero-Rodriguez María Cristina Romero-Rodriguez Nieves eAbril Rosa eSánchez-Lucas Jesus V eJorrin Novo |
author_sort |
María Cristina Romero-Rodriguez |
title |
Multiplex staining of 2-DE gels for an initial phosphoproteome analysis of germinating seeds and early grown seedlings from a non-orthodox spp.: Quercus ilex L. subsp. ballota [Desf.] Samp. |
title_short |
Multiplex staining of 2-DE gels for an initial phosphoproteome analysis of germinating seeds and early grown seedlings from a non-orthodox spp.: Quercus ilex L. subsp. ballota [Desf.] Samp. |
title_full |
Multiplex staining of 2-DE gels for an initial phosphoproteome analysis of germinating seeds and early grown seedlings from a non-orthodox spp.: Quercus ilex L. subsp. ballota [Desf.] Samp. |
title_fullStr |
Multiplex staining of 2-DE gels for an initial phosphoproteome analysis of germinating seeds and early grown seedlings from a non-orthodox spp.: Quercus ilex L. subsp. ballota [Desf.] Samp. |
title_full_unstemmed |
Multiplex staining of 2-DE gels for an initial phosphoproteome analysis of germinating seeds and early grown seedlings from a non-orthodox spp.: Quercus ilex L. subsp. ballota [Desf.] Samp. |
title_sort |
multiplex staining of 2-de gels for an initial phosphoproteome analysis of germinating seeds and early grown seedlings from a non-orthodox spp.: quercus ilex l. subsp. ballota [desf.] samp. |
publisher |
Frontiers Media S.A. |
series |
Frontiers in Plant Science |
issn |
1664-462X |
publishDate |
2015-08-01 |
description |
As a preliminary step in the phosphoproteome analysis of germinating seeds (0, and 24 h after seed imbibition) and early grown seedlings (216 h after seed imbibition) from a non-orthodox spp. Quercus ilex, a multiplex (SYPRO-Ruby and Pro-Q DPS) staining of high-resolution 2-DE gels was used. By using this protocol it was possible to detect changes in protein-abundance and/or phosphorylation status. This simple approach could be a good complementary alternative to the enrichment protocols used in the search for phosphoprotein candidates. While 482 spots were visualized with SYPRO-Ruby, 222 were with Pro-Q DPS. Statistically significant differences in spot intensity were observed among samples, these corresponding to 85 SYPRO-Ruby-, 20 Pro-Q-DPS-, and 35 SYPRO-Ruby and Pro-Q-DPS-stained spots. Fifty-five phosphoprotein candidates showing qualitative or quantitative differences between samples were subjected to MALDI-TOF-TOF MS analysis, with 20 of them being identified. Identified proteins belonged to five different functional categories, namely: carbohydrate and amino acid metabolism, defence, protein folding and oxidation-reduction processes. With the exception of a putative cyclase, the other nineteen proteins had at least one orthologous phosphoprotein in A. thaliana, M. truncatula, N. tabacum and G. max. Out of the twenty identified, seven showed differences in intensity in Pro-Q-DPS but not in SYPRO-Ruby-stained gels, including enzymes of the glycolysis and amino acid metabolism. This bears out that theory the regulation of these enzymes occurs at the post-translational level by phosphorylation with no changes at the transcriptional or translational level. This is different from the mechanism reported in orthodox seeds, in which concomitant changes in abundance and phosphorylation status have been observed for these enzymes. |
topic |
Germination phosphoproteomics post translational modification recalcitrant seeds Holm oak |
url |
http://journal.frontiersin.org/Journal/10.3389/fpls.2015.00620/full |
work_keys_str_mv |
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