Multiplex staining of 2-DE gels for an initial phosphoproteome analysis of germinating seeds and early grown seedlings from a non-orthodox specie: Quercus ilex L. subsp. ballota [Desf.] Samp.
Identifieur interne : 000078 ( Pmc/Corpus ); précédent : 000077; suivant : 000079Multiplex staining of 2-DE gels for an initial phosphoproteome analysis of germinating seeds and early grown seedlings from a non-orthodox specie: Quercus ilex L. subsp. ballota [Desf.] Samp.
Auteurs : M. Cristina Romero-Rodríguez ; Nieves Abril ; Rosa Sánchez-Lucas ; Jesús V. Jorrín-NovoSource :
- Frontiers in Plant Science [ 1664-462X ] ; 2015.
Abstract
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 sp.
Url:
DOI: 10.3389/fpls.2015.00620
PubMed: 26322061
PubMed Central: 4531236
Links to Exploration step
PMC:4531236Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Multiplex staining of 2-DE gels for an initial phosphoproteome analysis of germinating seeds and early grown seedlings from a non-orthodox specie: <italic>Quercus ilex</italic> L. subsp. <italic>ballota</italic> [Desf.] Samp.</title><author><name sortKey="Romero Rodriguez, M Cristina" sort="Romero Rodriguez, M Cristina" uniqKey="Romero Rodriguez M" first="M. Cristina" last="Romero-Rodríguez">M. Cristina Romero-Rodríguez</name><affiliation><nlm:aff id="aff1"><institution>Department of Biochemistry and Molecular Biology, University of Cordoba</institution><country>Cordoba, Spain</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff2"><institution>Agricultural and Plant Proteomics Research Group, Department of Biochemistry and Molecular Biology, Escuela Técnica Superior de Ingenieros Agrónomos y de Montes, University of Cordoba</institution><country>Cordoba, Spain</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff3"><institution>Centro Multidisciplinario de Investigaciones Tecnológicas, Universidad Nacional de Asunción</institution><country>San Lorenzo, Paraguay</country></nlm:aff></affiliation></author><author><name sortKey="Abril, Nieves" sort="Abril, Nieves" uniqKey="Abril N" first="Nieves" last="Abril">Nieves Abril</name><affiliation><nlm:aff id="aff1"><institution>Department of Biochemistry and Molecular Biology, University of Cordoba</institution><country>Cordoba, Spain</country></nlm:aff></affiliation></author><author><name sortKey="Sanchez Lucas, Rosa" sort="Sanchez Lucas, Rosa" uniqKey="Sanchez Lucas R" first="Rosa" last="Sánchez-Lucas">Rosa Sánchez-Lucas</name><affiliation><nlm:aff id="aff1"><institution>Department of Biochemistry and Molecular Biology, University of Cordoba</institution><country>Cordoba, Spain</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff2"><institution>Agricultural and Plant Proteomics Research Group, Department of Biochemistry and Molecular Biology, Escuela Técnica Superior de Ingenieros Agrónomos y de Montes, University of Cordoba</institution><country>Cordoba, Spain</country></nlm:aff></affiliation></author><author><name sortKey="Jorrin Novo, Jesus V" sort="Jorrin Novo, Jesus V" uniqKey="Jorrin Novo J" first="Jesús V." last="Jorrín-Novo">Jesús V. Jorrín-Novo</name><affiliation><nlm:aff id="aff1"><institution>Department of Biochemistry and Molecular Biology, University of Cordoba</institution><country>Cordoba, Spain</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff2"><institution>Agricultural and Plant Proteomics Research Group, Department of Biochemistry and Molecular Biology, Escuela Técnica Superior de Ingenieros Agrónomos y de Montes, University of Cordoba</institution><country>Cordoba, Spain</country></nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">26322061</idno><idno type="pmc">4531236</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4531236</idno><idno type="RBID">PMC:4531236</idno><idno type="doi">10.3389/fpls.2015.00620</idno><date when="2015">2015</date><idno type="wicri:Area/Pmc/Corpus">000078</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Multiplex staining of 2-DE gels for an initial phosphoproteome analysis of germinating seeds and early grown seedlings from a non-orthodox specie: <italic>Quercus ilex</italic> L. subsp. <italic>ballota</italic> [Desf.] Samp.</title><author><name sortKey="Romero Rodriguez, M Cristina" sort="Romero Rodriguez, M Cristina" uniqKey="Romero Rodriguez M" first="M. Cristina" last="Romero-Rodríguez">M. Cristina Romero-Rodríguez</name><affiliation><nlm:aff id="aff1"><institution>Department of Biochemistry and Molecular Biology, University of Cordoba</institution><country>Cordoba, Spain</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff2"><institution>Agricultural and Plant Proteomics Research Group, Department of Biochemistry and Molecular Biology, Escuela Técnica Superior de Ingenieros Agrónomos y de Montes, University of Cordoba</institution><country>Cordoba, Spain</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff3"><institution>Centro Multidisciplinario de Investigaciones Tecnológicas, Universidad Nacional de Asunción</institution><country>San Lorenzo, Paraguay</country></nlm:aff></affiliation></author><author><name sortKey="Abril, Nieves" sort="Abril, Nieves" uniqKey="Abril N" first="Nieves" last="Abril">Nieves Abril</name><affiliation><nlm:aff id="aff1"><institution>Department of Biochemistry and Molecular Biology, University of Cordoba</institution><country>Cordoba, Spain</country></nlm:aff></affiliation></author><author><name sortKey="Sanchez Lucas, Rosa" sort="Sanchez Lucas, Rosa" uniqKey="Sanchez Lucas R" first="Rosa" last="Sánchez-Lucas">Rosa Sánchez-Lucas</name><affiliation><nlm:aff id="aff1"><institution>Department of Biochemistry and Molecular Biology, University of Cordoba</institution><country>Cordoba, Spain</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff2"><institution>Agricultural and Plant Proteomics Research Group, Department of Biochemistry and Molecular Biology, Escuela Técnica Superior de Ingenieros Agrónomos y de Montes, University of Cordoba</institution><country>Cordoba, Spain</country></nlm:aff></affiliation></author><author><name sortKey="Jorrin Novo, Jesus V" sort="Jorrin Novo, Jesus V" uniqKey="Jorrin Novo J" first="Jesús V." last="Jorrín-Novo">Jesús V. Jorrín-Novo</name><affiliation><nlm:aff id="aff1"><institution>Department of Biochemistry and Molecular Biology, University of Cordoba</institution><country>Cordoba, Spain</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff2"><institution>Agricultural and Plant Proteomics Research Group, Department of Biochemistry and Molecular Biology, Escuela Técnica Superior de Ingenieros Agrónomos y de Montes, University of Cordoba</institution><country>Cordoba, Spain</country></nlm:aff></affiliation></author></analytic><series><title level="j">Frontiers in Plant Science</title><idno type="eISSN">1664-462X</idno><imprint><date when="2015">2015</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>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 sp. <italic>Quercus ilex</italic>, 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, defense, protein folding, and oxidation-reduction processes. With the exception of a putative cyclase, the other 19 proteins had at least one orthologous phosphoprotein in <italic>Arabidopsis thaliana, Medicago truncatula, N. tabacum</italic>, and <italic>Glycine max</italic>. Out of the 20 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.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Agrawal, G K" uniqKey="Agrawal G">G. K. Agrawal</name></author><author><name sortKey="Thelen, J J" uniqKey="Thelen J">J. J. Thelen</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Agrawal, G K" uniqKey="Agrawal G">G. K. Agrawal</name></author><author><name sortKey="Thelen, J J" uniqKey="Thelen J">J. J. Thelen</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Alonso, J" uniqKey="Alonso J">J. Alonso</name></author><author><name sortKey="Zapata, C" uniqKey="Zapata C">C. Zapata</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Balbuena, T S" uniqKey="Balbuena T">T. S. Balbuena</name></author><author><name sortKey="Jo, L" uniqKey="Jo L">L. Jo</name></author><author><name sortKey="Pieruzzi, F P" uniqKey="Pieruzzi F">F. P. Pieruzzi</name></author><author><name sortKey="Dias, L L C" uniqKey="Dias L">L. L. C. Dias</name></author><author><name sortKey="Silveira, V" uniqKey="Silveira V">V. Silveira</name></author><author><name sortKey="Santa Catarina, C" uniqKey="Santa Catarina C">C. Santa-Catarina</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Bao, J" uniqKey="Bao J">J. Bao</name></author><author><name sortKey="Cheung, W Y" uniqKey="Cheung W">W. Y. Cheung</name></author><author><name sortKey="Jy, W" uniqKey="Jy W">W Jy</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Berggren, K" uniqKey="Berggren K">K. Berggren</name></author><author><name sortKey="Chernokalskaya, E" uniqKey="Chernokalskaya E">E. Chernokalskaya</name></author><author><name sortKey="Steinberg, T H" uniqKey="Steinberg T">T. H. Steinberg</name></author><author><name sortKey="Kemper, C" uniqKey="Kemper C">C. Kemper</name></author><author><name sortKey="Lopez, M F" uniqKey="Lopez M">M. F. Lopez</name></author><author><name sortKey="Diwu, Z" uniqKey="Diwu Z">Z. Diwu</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Bond, A E" uniqKey="Bond A">A. E. Bond</name></author><author><name sortKey="Row, P E" uniqKey="Row P">P. E. Row</name></author><author><name sortKey="Dudley, E" uniqKey="Dudley E">E. Dudley</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Bradford, M M" uniqKey="Bradford M">M. M. Bradford</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Chen, F" uniqKey="Chen F">F. Chen</name></author><author><name sortKey="Jiang, L" uniqKey="Jiang L">L. Jiang</name></author><author><name sortKey="Zheng, J" uniqKey="Zheng J">J. Zheng</name></author><author><name sortKey="Huang, R" uniqKey="Huang R">R. Huang</name></author><author><name sortKey="Wang, H" uniqKey="Wang H">H. Wang</name></author><author><name sortKey="Hong, Z" uniqKey="Hong Z">Z. Hong</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Conesa, A" uniqKey="Conesa A">A. Conesa</name></author><author><name sortKey="Gotz, S" uniqKey="Gotz S">S. Götz</name></author><author><name sortKey="Garcia G Mez, J M" uniqKey="Garcia G Mez J">J. M. García-Gómez</name></author><author><name sortKey="Terol, J" uniqKey="Terol J">J. Terol</name></author><author><name sortKey="Tal N, M" uniqKey="Tal N M">M. Talón</name></author><author><name sortKey="Robles, M" uniqKey="Robles M">M. Robles</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Cutler, S R" uniqKey="Cutler S">S. R. Cutler</name></author><author><name sortKey="Rodriguez, P L" uniqKey="Rodriguez P">P. L. Rodriguez</name></author><author><name sortKey="Finkelstein, R R" uniqKey="Finkelstein R">R. R. Finkelstein</name></author><author><name sortKey="Abrams, S R" uniqKey="Abrams S">S. R. Abrams</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Fila, J" uniqKey="Fila J">J. Fíla</name></author><author><name sortKey="Matros, A" uniqKey="Matros A">A. Matros</name></author><author><name sortKey="Radau, S" uniqKey="Radau S">S. Radau</name></author><author><name sortKey="Zahedi, R P" uniqKey="Zahedi R">R. P. Zahedi</name></author><author><name sortKey="C Apkova, V" uniqKey="C Apkova V">V. C̀apková</name></author><author><name sortKey="Mock, H P" uniqKey="Mock H">H.-P. Mock</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Fujii, H" uniqKey="Fujii H">H. Fujii</name></author><author><name sortKey="Chinnusamy, V" uniqKey="Chinnusamy V">V. Chinnusamy</name></author><author><name sortKey="Rodrigues, A" uniqKey="Rodrigues A">A. Rodrigues</name></author><author><name sortKey="Rubio, S" uniqKey="Rubio S">S. Rubio</name></author><author><name sortKey="Antoni, R" uniqKey="Antoni R">R. Antoni</name></author><author><name sortKey="Park, S Y" uniqKey="Park S">S.-Y. Park</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Gallego, F J" uniqKey="Gallego F">F. J. Gallego</name></author><author><name sortKey="De Algaba, A P" uniqKey="De Algaba A">A. P. De Algaba</name></author><author><name sortKey="Fernandez Escobar, R" uniqKey="Fernandez Escobar R">R. Fernandez-Escobar</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Gao, J" uniqKey="Gao J">J. Gao</name></author><author><name sortKey="Agrawal, G K" uniqKey="Agrawal G">G. K. Agrawal</name></author><author><name sortKey="Thelen, J J" uniqKey="Thelen J">J. J. Thelen</name></author><author><name sortKey="Xu, D" uniqKey="Xu D">D. Xu</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Gorg, A" uniqKey="Gorg A">A. Görg</name></author><author><name sortKey="Weiss, W" uniqKey="Weiss W">W. Weiss</name></author><author><name sortKey="Dunn, M J" uniqKey="Dunn M">M. J. Dunn</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Grimsrud, P A" uniqKey="Grimsrud P">P. A. Grimsrud</name></author><author><name sortKey="Den Os, D" uniqKey="Den Os D">D. Den Os</name></author><author><name sortKey="Wenger, C D" uniqKey="Wenger C">C. D. Wenger</name></author><author><name sortKey="Swaney, D L" uniqKey="Swaney D">D. L. Swaney</name></author><author><name sortKey="Schwartz, D" uniqKey="Schwartz D">D. Schwartz</name></author><author><name sortKey="Sussman, M R" uniqKey="Sussman M">M. R. Sussman</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Han, C" uniqKey="Han C">C. Han</name></author><author><name sortKey="Wang, K" uniqKey="Wang K">K. Wang</name></author><author><name sortKey="Yang, P" uniqKey="Yang P">P. Yang</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Hunt, L" uniqKey="Hunt L">L. Hunt</name></author><author><name sortKey="Holdsworth, M J" uniqKey="Holdsworth M">M. J. Holdsworth</name></author><author><name sortKey="Gray, J E" uniqKey="Gray J">J. E. Gray</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Jones, A M E" uniqKey="Jones A">A. M. E. Jones</name></author><author><name sortKey="Maclean, D" uniqKey="Maclean D">D. Maclean</name></author><author><name sortKey="Studholme, D J" uniqKey="Studholme D">D. J. Studholme</name></author><author><name sortKey="Serna Sanz, A" uniqKey="Serna Sanz A">A. Serna-Sanz</name></author><author><name sortKey="Andreasson, E" uniqKey="Andreasson E">E. Andreasson</name></author><author><name sortKey="Rathjen, J P" uniqKey="Rathjen J">J. P. Rathjen</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Jorge, I" uniqKey="Jorge I">I. Jorge</name></author><author><name sortKey="Navarro, R M" uniqKey="Navarro R">R. M. Navarro</name></author><author><name sortKey="Lenz, C" uniqKey="Lenz C">C. Lenz</name></author><author><name sortKey="Ariza, D" uniqKey="Ariza D">D. Ariza</name></author><author><name sortKey="Jorrin, J" uniqKey="Jorrin J">J. Jorrín</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kaufmann, H" uniqKey="Kaufmann H">H. Kaufmann</name></author><author><name sortKey="Bailey, J E" uniqKey="Bailey J">J. E. Bailey</name></author><author><name sortKey="Fussenegger, M" uniqKey="Fussenegger M">M. Fussenegger</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kersten, B" uniqKey="Kersten B">B. Kersten</name></author><author><name sortKey="Agrawal, G K" uniqKey="Agrawal G">G. K. Agrawal</name></author><author><name sortKey="Durek, P" uniqKey="Durek P">P. Durek</name></author><author><name sortKey="Neigenfind, J" uniqKey="Neigenfind J">J. Neigenfind</name></author><author><name sortKey="Schulze, W" uniqKey="Schulze W">W. Schulze</name></author><author><name sortKey="Walther, D" uniqKey="Walther D">D. Walther</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kersten, B" uniqKey="Kersten B">B. Kersten</name></author><author><name sortKey="Agrawal, G K" uniqKey="Agrawal G">G. K. Agrawal</name></author><author><name sortKey="Iwahashi, H" uniqKey="Iwahashi H">H. Iwahashi</name></author><author><name sortKey="Rakwal, R" uniqKey="Rakwal R">R. Rakwal</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Laemmli, U K" uniqKey="Laemmli U">U. K. Laemmli</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Leitner, A" uniqKey="Leitner A">A. Leitner</name></author><author><name sortKey="Sturm, M" uniqKey="Sturm M">M. Sturm</name></author><author><name sortKey="Lindner, W" uniqKey="Lindner W">W. Lindner</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Li, H" uniqKey="Li H">H. Li</name></author><author><name sortKey="Wong, W S" uniqKey="Wong W">W. S. Wong</name></author><author><name sortKey="Zhu, L" uniqKey="Zhu L">L. Zhu</name></author><author><name sortKey="Guo, H W" uniqKey="Guo H">H. W. Guo</name></author><author><name sortKey="Ecker, J" uniqKey="Ecker J">J. Ecker</name></author><author><name sortKey="Li, N" uniqKey="Li N">N. Li</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Li, J" uniqKey="Li J">J. Li</name></author><author><name sortKey="Silva Sanchez, C" uniqKey="Silva Sanchez C">C. Silva-Sanchez</name></author><author><name sortKey="Zhang, T" uniqKey="Zhang T">T. Zhang</name></author><author><name sortKey="Chen, S" uniqKey="Chen S">S. Chen</name></author><author><name sortKey="Li, H" uniqKey="Li H">H. Li</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Li, X" uniqKey="Li X">X. Li</name></author><author><name sortKey="Zhuo, J" uniqKey="Zhuo J">J. Zhuo</name></author><author><name sortKey="Jing, Y" uniqKey="Jing Y">Y. Jing</name></author><author><name sortKey="Liu, X" uniqKey="Liu X">X. Liu</name></author><author><name sortKey="Wang, X" uniqKey="Wang X">X. Wang</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Liu, Y" uniqKey="Liu Y">Y. Liu</name></author><author><name sortKey="Liu, G" uniqKey="Liu G">G. Liu</name></author><author><name sortKey="Li, Q" uniqKey="Li Q">Q. Li</name></author><author><name sortKey="Liu, Y" uniqKey="Liu Y">Y. Liu</name></author><author><name sortKey="Hou, L" uniqKey="Hou L">L. Hou</name></author><author><name sortKey="Li, G" uniqKey="Li G">G. Li</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lu, T C" uniqKey="Lu T">T.-C. Lu</name></author><author><name sortKey="Meng, L B" uniqKey="Meng L">L.-B. Meng</name></author><author><name sortKey="Yang, C P" uniqKey="Yang C">C.-P. Yang</name></author><author><name sortKey="Liu, G F" uniqKey="Liu G">G.-F. Liu</name></author><author><name sortKey="Liu, G J" uniqKey="Liu G">G.-J. Liu</name></author><author><name sortKey="Ma, W" uniqKey="Ma W">W. Ma</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Maldonado, A M" uniqKey="Maldonado A">A. M. Maldonado</name></author><author><name sortKey="Echevarria Zome O, S" uniqKey="Echevarria Zome O S">S. Echevarría-Zomeño</name></author><author><name sortKey="Jean Baptiste, S" uniqKey="Jean Baptiste S">S. Jean-Baptiste</name></author><author><name sortKey="Hernandez, M" uniqKey="Hernandez M">M. Hernández</name></author><author><name sortKey="Jorrin Novo, J V" uniqKey="Jorrin Novo J">J. V. Jorrín-Novo</name></author></analytic></biblStruct><biblStruct></biblStruct><biblStruct><analytic><author><name sortKey="Meyer, L J" uniqKey="Meyer L">L. J. Meyer</name></author><author><name sortKey="Gao, J" uniqKey="Gao J">J. Gao</name></author><author><name sortKey="Xu, D" uniqKey="Xu D">D. Xu</name></author><author><name sortKey="Thelen, J J" uniqKey="Thelen J">J. J. Thelen</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Nakagami, H" uniqKey="Nakagami H">H. Nakagami</name></author><author><name sortKey="Sugiyama, N" uniqKey="Sugiyama N">N. Sugiyama</name></author><author><name sortKey="Mochida, K" uniqKey="Mochida K">K. Mochida</name></author><author><name sortKey="Daudi, A" uniqKey="Daudi A">A. Daudi</name></author><author><name sortKey="Yoshida, Y" uniqKey="Yoshida Y">Y. Yoshida</name></author><author><name sortKey="Toyoda, T" uniqKey="Toyoda T">T. Toyoda</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Pulido, F J" uniqKey="Pulido F">F. J. Pulido</name></author><author><name sortKey="Diaz, M" uniqKey="Diaz M">M. DíAz</name></author><author><name sortKey="Hidalgo De Trucios, S J" uniqKey="Hidalgo De Trucios S">S. J. Hidalgo De Trucios</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Reiland, S" uniqKey="Reiland S">S. Reiland</name></author><author><name sortKey="Messerli, G" uniqKey="Messerli G">G. Messerli</name></author><author><name sortKey="Baerenfaller, K" uniqKey="Baerenfaller K">K. Baerenfaller</name></author><author><name sortKey="Gerrits, B" uniqKey="Gerrits B">B. Gerrits</name></author><author><name sortKey="Endler, A" uniqKey="Endler A">A. Endler</name></author><author><name sortKey="Grossmann, J" uniqKey="Grossmann J">J. Grossmann</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Rigbolt, K T G" uniqKey="Rigbolt K">K. T. G. Rigbolt</name></author><author><name sortKey="Blagoev, B" uniqKey="Blagoev B">B. Blagoev</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Romero Rodriguez, M C" uniqKey="Romero Rodriguez M">M. C. Romero-Rodríguez</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Romero Rodriguez, M C" uniqKey="Romero Rodriguez M">M. C. Romero-Rodriguez</name></author><author><name sortKey="Pascual, J" uniqKey="Pascual J">J. Pascual</name></author><author><name sortKey="Valledor, L" uniqKey="Valledor L">L. Valledor</name></author><author><name sortKey="Jorrin Novo, J" uniqKey="Jorrin Novo J">J. Jorrin-Novo</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Rose, C M" uniqKey="Rose C">C. M. Rose</name></author><author><name sortKey="Venkateshwaran, M" uniqKey="Venkateshwaran M">M. Venkateshwaran</name></author><author><name sortKey="Grimsrud, P A" uniqKey="Grimsrud P">P. A. Grimsrud</name></author><author><name sortKey="Westphall, M S" uniqKey="Westphall M">M. S. Westphall</name></author><author><name sortKey="Sussman, M R" uniqKey="Sussman M">M. R. Sussman</name></author><author><name sortKey="Coon, J J" uniqKey="Coon J">J. J. Coon</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Rose, C M" uniqKey="Rose C">C. M. Rose</name></author><author><name sortKey="Venkateshwaran, M" uniqKey="Venkateshwaran M">M. Venkateshwaran</name></author><author><name sortKey="Volkening, J D" uniqKey="Volkening J">J. D. Volkening</name></author><author><name sortKey="Grimsrud, P A" uniqKey="Grimsrud P">P. A. Grimsrud</name></author><author><name sortKey="Maeda, J" uniqKey="Maeda J">J. Maeda</name></author><author><name sortKey="Bailey, D J" uniqKey="Bailey D">D. J. Bailey</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Sghaier Hammami, B" uniqKey="Sghaier Hammami B">B. Sghaier-Hammami</name></author><author><name sortKey="Valero Galvan, J" uniqKey="Valero Galvan J">J. Valero-Galvàn</name></author><author><name sortKey="Romero Rodriguez, M C" uniqKey="Romero Rodriguez M">M. C. Romero-Rodríguez</name></author><author><name sortKey="Navarro Cerrillo, R M" uniqKey="Navarro Cerrillo R">R. M. Navarro-Cerrillo</name></author><author><name sortKey="Abdelly, C" uniqKey="Abdelly C">C. Abdelly</name></author><author><name sortKey="Jorrin Novo, J" uniqKey="Jorrin Novo J">J. Jorrín-Novo</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Sharov, A A" uniqKey="Sharov A">A. A. Sharov</name></author><author><name sortKey="Dudekula, D B" uniqKey="Dudekula D">D. B. Dudekula</name></author><author><name sortKey="Ko, M S H" uniqKey="Ko M">M. S. H. Ko</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Shevchenko, A" uniqKey="Shevchenko A">A. Shevchenko</name></author><author><name sortKey="Wilm, M" uniqKey="Wilm M">M. Wilm</name></author><author><name sortKey="Vorm, O" uniqKey="Vorm O">O. Vorm</name></author><author><name sortKey="Mann, M" uniqKey="Mann M">M. Mann</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Subba, P" uniqKey="Subba P">P. Subba</name></author><author><name sortKey="Barua, P" uniqKey="Barua P">P. Barua</name></author><author><name sortKey="Kumar, R" uniqKey="Kumar R">R. Kumar</name></author><author><name sortKey="Datta, A" uniqKey="Datta A">A. Datta</name></author><author><name sortKey="Soni, K K" uniqKey="Soni K">K. K. Soni</name></author><author><name sortKey="Chakraborty, S" uniqKey="Chakraborty S">S. Chakraborty</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Sugiyama, N" uniqKey="Sugiyama N">N. Sugiyama</name></author><author><name sortKey="Nakagami, H" uniqKey="Nakagami H">H. Nakagami</name></author><author><name sortKey="Mochida, K" uniqKey="Mochida K">K. Mochida</name></author><author><name sortKey="Daudi, A" uniqKey="Daudi A">A. Daudi</name></author><author><name sortKey="Tomita, M" uniqKey="Tomita M">M. Tomita</name></author><author><name sortKey="Shirasu, K" uniqKey="Shirasu K">K. Shirasu</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Taji, T" uniqKey="Taji T">T. Taji</name></author><author><name sortKey="Ohsumi, C" uniqKey="Ohsumi C">C. Ohsumi</name></author><author><name sortKey="Iuchi, S" uniqKey="Iuchi S">S. Iuchi</name></author><author><name sortKey="Seki, M" uniqKey="Seki M">M. Seki</name></author><author><name sortKey="Kasuga, M" uniqKey="Kasuga M">M. Kasuga</name></author><author><name sortKey="Kobayashi, M" uniqKey="Kobayashi M">M. Kobayashi</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Thingholm, T E" uniqKey="Thingholm T">T. E. Thingholm</name></author><author><name sortKey="Jensen, O N" uniqKey="Jensen O">O. N. Jensen</name></author><author><name sortKey="Larsen, M R" uniqKey="Larsen M">M. R. Larsen</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Umezawa, T" uniqKey="Umezawa T">T. Umezawa</name></author><author><name sortKey="Sugiyama, N" uniqKey="Sugiyama N">N. Sugiyama</name></author><author><name sortKey="Takahashi, F" uniqKey="Takahashi F">F. Takahashi</name></author><author><name sortKey="Anderson, J C" uniqKey="Anderson J">J. C. Anderson</name></author><author><name sortKey="Ishihama, Y" uniqKey="Ishihama Y">Y. Ishihama</name></author><author><name sortKey="Peck, S C" uniqKey="Peck S">S. C. Peck</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Valero Galvan, J" uniqKey="Valero Galvan J">J. Valero Galván</name></author><author><name sortKey="Jorrin Novo, J" uniqKey="Jorrin Novo J">J. Jorrín Novo</name></author><author><name sortKey="Cabrera, A" uniqKey="Cabrera A">A. Cabrera</name></author><author><name sortKey="Ariza, D" uniqKey="Ariza D">D. Ariza</name></author><author><name sortKey="Garcia Olmo, J" uniqKey="Garcia Olmo J">J. García-Olmo</name></author><author><name sortKey="Cerrillo, R" uniqKey="Cerrillo R">R. Cerrillo</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Valero Galvan, J" uniqKey="Valero Galvan J">J. Valero Galván</name></author><author><name sortKey="Valledor, L" uniqKey="Valledor L">L. Valledor</name></author><author><name sortKey="Cerrillo, R M N" uniqKey="Cerrillo R">R. M. N. Cerrillo</name></author><author><name sortKey="Pelegrin, E G" uniqKey="Pelegrin E">E. G. Pelegrín</name></author><author><name sortKey="Jorrin Novo, J V" uniqKey="Jorrin Novo J">J. V. Jorrín-Novo</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Valero Galvan, J" uniqKey="Valero Galvan J">J. Valero Galván</name></author><author><name sortKey="Valledor, L" uniqKey="Valledor L">L. Valledor</name></author><author><name sortKey="Gonzalez Fernandez, R" uniqKey="Gonzalez Fernandez R">R. González Fernandez</name></author><author><name sortKey="Navarro Cerrillo, R M" uniqKey="Navarro Cerrillo R">R. M. Navarro Cerrillo</name></author><author><name sortKey="Jorrin Novo, J V" uniqKey="Jorrin Novo J">J. V. Jorrín-Novo</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Valledor, L" uniqKey="Valledor L">L. Valledor</name></author><author><name sortKey="Jorrin, J" uniqKey="Jorrin J">J. Jorrín</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Walters, C" uniqKey="Walters C">C. Walters</name></author><author><name sortKey="Berjak, P" uniqKey="Berjak P">P. Berjak</name></author><author><name sortKey="Pammenter, N" uniqKey="Pammenter N">N. Pammenter</name></author><author><name sortKey="Kennedy, K" uniqKey="Kennedy K">K. Kennedy</name></author><author><name sortKey="Raven, P" uniqKey="Raven P">P. Raven</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Wang, W" uniqKey="Wang W">W. Wang</name></author><author><name sortKey="Vignani, R" uniqKey="Vignani R">R. Vignani</name></author><author><name sortKey="Scali, M" uniqKey="Scali M">M. Scali</name></author><author><name sortKey="Cresti, M" uniqKey="Cresti M">M. Cresti</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Woods Ignatoski, K" uniqKey="Woods Ignatoski K">K. Woods Ignatoski</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Wu, R" uniqKey="Wu R">R. Wu</name></author><author><name sortKey="Haas, W" uniqKey="Haas W">W. Haas</name></author><author><name sortKey="Dephoure, N" uniqKey="Dephoure N">N. Dephoure</name></author><author><name sortKey="Huttlin, E L" uniqKey="Huttlin E">E. L. Huttlin</name></author><author><name sortKey="Zhai, B" uniqKey="Zhai B">B. Zhai</name></author><author><name sortKey="Sowa, M E" uniqKey="Sowa M">M. E. Sowa</name></author></analytic></biblStruct></listBibl></div1></back></TEI><pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">Front Plant Sci</journal-id><journal-id journal-id-type="iso-abbrev">Front Plant Sci</journal-id><journal-id journal-id-type="publisher-id">Front. Plant Sci.</journal-id><journal-title-group><journal-title>Frontiers in Plant Science</journal-title></journal-title-group><issn pub-type="epub">1664-462X</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">26322061</article-id><article-id pub-id-type="pmc">4531236</article-id><article-id pub-id-type="doi">10.3389/fpls.2015.00620</article-id><article-categories><subj-group subj-group-type="heading"><subject>Plant Science</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>Multiplex staining of 2-DE gels for an initial phosphoproteome analysis of germinating seeds and early grown seedlings from a non-orthodox specie: <italic>Quercus ilex</italic> L. subsp. <italic>ballota</italic> [Desf.] Samp.</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Romero-Rodríguez</surname><given-names>M. Cristina</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="aff" rid="aff2"><sup>2</sup></xref><xref ref-type="aff" rid="aff3"><sup>3</sup></xref><uri xlink:type="simple" xlink:href="http://loop.frontiersin.org/people/222716/overview"></uri></contrib><contrib contrib-type="author"><name><surname>Abril</surname><given-names>Nieves</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><uri xlink:type="simple" xlink:href="http://loop.frontiersin.org/people/228320/overview"></uri></contrib><contrib contrib-type="author"><name><surname>Sánchez-Lucas</surname><given-names>Rosa</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="aff" rid="aff2"><sup>2</sup></xref><uri xlink:type="simple" xlink:href="http://loop.frontiersin.org/people/234640/overview"></uri></contrib><contrib contrib-type="author"><name><surname>Jorrín-Novo</surname><given-names>Jesús V.</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="aff" rid="aff2"><sup>2</sup></xref><xref ref-type="author-notes" rid="fn001"><sup>*</sup></xref><uri xlink:type="simple" xlink:href="http://loop.frontiersin.org/people/40741/overview"></uri></contrib></contrib-group><aff id="aff1"><sup>1</sup><institution>Department of Biochemistry and Molecular Biology, University of Cordoba</institution><country>Cordoba, Spain</country></aff><aff id="aff2"><sup>2</sup><institution>Agricultural and Plant Proteomics Research Group, Department of Biochemistry and Molecular Biology, Escuela Técnica Superior de Ingenieros Agrónomos y de Montes, University of Cordoba</institution><country>Cordoba, Spain</country></aff><aff id="aff3"><sup>3</sup><institution>Centro Multidisciplinario de Investigaciones Tecnológicas, Universidad Nacional de Asunción</institution><country>San Lorenzo, Paraguay</country></aff><author-notes><fn fn-type="edited-by"><p>Edited by: Nicolas L. Taylor, The University of Western Australia, Australia</p></fn><fn fn-type="edited-by"><p>Reviewed by: Martin Hajduch, Slovak Academy of Sciences, Slovakia; Deyou Qiu, Chinese Academy of Forestry, China</p></fn><corresp id="fn001">*Correspondence: Jesús V. Jorrín-Novo, Department of Biochemistry and Molecular Biology, University of Cordoba, Campus de Rabanales, Ed. Severo Ochoa, Planta Baja, 14071 Cordoba, Spain <email xlink:type="simple">bf1jonoj@uco.es</email></corresp><fn fn-type="other" id="fn002"><p>This article was submitted to Plant Proteomics, a section of the journal Frontiers in Plant Science</p></fn></author-notes><pub-date pub-type="epub"><day>11</day><month>8</month><year>2015</year></pub-date><pub-date pub-type="collection"><year>2015</year></pub-date><volume>6</volume><elocation-id>620</elocation-id><history><date date-type="received"><day>07</day><month>4</month><year>2015</year></date><date date-type="accepted"><day>27</day><month>7</month><year>2015</year></date></history><permissions><copyright-statement>Copyright © 2015 Romero-Rodríguez, Abril, Sánchez-Lucas and Jorrín-Novo.</copyright-statement><copyright-year>2015</copyright-year><copyright-holder>Romero-Rodríguez, Abril, Sánchez-Lucas and Jorrín-Novo</copyright-holder><license xlink:href="http://creativecommons.org/licenses/by/4.0/"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>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 sp. <italic>Quercus ilex</italic>, 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, defense, protein folding, and oxidation-reduction processes. With the exception of a putative cyclase, the other 19 proteins had at least one orthologous phosphoprotein in <italic>Arabidopsis thaliana, Medicago truncatula, N. tabacum</italic>, and <italic>Glycine max</italic>. Out of the 20 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.</p></abstract><kwd-group><kwd>holm oak</kwd><kwd>recalcitrant seeds</kwd><kwd>germination</kwd><kwd>phosphoproteomics</kwd><kwd>post translational modification</kwd></kwd-group><counts><fig-count count="3"></fig-count><table-count count="3"></table-count><equation-count count="0"></equation-count><ref-count count="58"></ref-count><page-count count="14"></page-count><word-count count="7132"></word-count></counts></article-meta></front><body><sec sec-type="intro" id="s1"><title>Introduction</title><p>Holm oak (<italic>Quercus ilex</italic> L. subsp. <italic>ballota</italic> [Desf.] Samp.) is the dominant tree species in natural forest ecosystems over large areas of the Western Mediterranean Basin (Pulido et al., <xref rid="B36" ref-type="bibr">2001</xref>). Nowadays, forest restoration and reforestation are high priority objectives, with <italic>Q. ilex</italic> being one of the major tree species for such a purpose (MAPA, <xref rid="B33" ref-type="bibr">2006</xref>), this requiring its nursery production at a high scale. <italic>Q. ilex</italic> forest maintenance and sustainability are facing important problems and challenges related to seed viability/conservation and mortality of adult trees and plantlets after field transplantation resulting from adverse environmental conditions like drought and the so-called decline syndrome (Gallego et al., <xref rid="B14" ref-type="bibr">1999</xref>). As natural, non-domesticated, plant species with a great plasticity and phenotypic variability, a key challenge prior to massive clonal propagation is the establishment of techniques for the cataloging and selection of genotypes among provenances with a high survival percentage and productivity under adverse environmental conditions. In our group, a Proteomics Research Program with <italic>Q. ilex</italic> has been carried out in order to study variability of holm oak populations and response to stresses, and to select elite individuals to be used in reforestation programmes (Jorge et al., <xref rid="B21" ref-type="bibr">2006</xref>; Valero Galván et al., <xref rid="B52" ref-type="bibr">2011</xref>, <xref rid="B51" ref-type="bibr">2012a</xref>,<xref rid="B53" ref-type="bibr">b</xref>).</p><p>Holm oak is a recalcitrant plant species whose germination and viability loss during storage, has been poorly studied at the molecular level if compared with to orthodox ones. This knowledge will help to understand biochemistry and metabolic status before and after the germination process, which could be important for the development and optimization of strategies for large scale propagation, germplasm conservation and seed conservation practices (Balbuena et al., <xref rid="B4" ref-type="bibr">2011</xref>; Walters et al., <xref rid="B55" ref-type="bibr">2013</xref>).</p><p>The germination process of plant seeds has been analyzed by using a proteomics approach both for comparative purposes and for characterisation of posttranslational modifications (PTMs), mainly phosphorylation. Phosphorylation is a ubiquitous and reversible PTM, which determines protein conformation, stability and activity (Kersten et al., <xref rid="B24" ref-type="bibr">2006</xref>; Hunt et al., <xref rid="B19" ref-type="bibr">2007</xref>; Bond et al., <xref rid="B7" ref-type="bibr">2011</xref>). Phosphorylation events modulate a wide range of biological processes in plants and other organisms (Nakagami et al., <xref rid="B35" ref-type="bibr">2010</xref>). Thus, in seed germination, phosphorylation has proven to be one of the mechanisms underlying the signaling cascade pathway mediated by ABA (Fujii et al., <xref rid="B13" ref-type="bibr">2009</xref>; Cutler et al., <xref rid="B11" ref-type="bibr">2010</xref>; Umezawa et al., <xref rid="B50" ref-type="bibr">2013</xref>). Quantitative and qualitative profiling of phosphoproteins during seed germination and seedling development has been performed using different proteomic approaches (gel based and gel-free) in different plant species such as <italic>Arabidopsis thaliana</italic> (Sugiyama et al., <xref rid="B47" ref-type="bibr">2008</xref>; Kersten et al., <xref rid="B23" ref-type="bibr">2009</xref>; Reiland et al., <xref rid="B37" ref-type="bibr">2009</xref>), <italic>Medicago truncatula</italic> (Kersten et al., <xref rid="B23" ref-type="bibr">2009</xref>; Rose et al., <xref rid="B41" ref-type="bibr">2012a</xref>), <italic>Phaseolus vulgaris</italic> (Alonso and Zapata, <xref rid="B3" ref-type="bibr">2014</xref>), <italic>Zea mays</italic> (Lu et al., <xref rid="B31" ref-type="bibr">2008</xref>), and <italic>Oryza sativa</italic> (Chen et al., <xref rid="B9" ref-type="bibr">2014</xref>; Han et al., <xref rid="B18" ref-type="bibr">2014</xref>). It is important to highlight that to the best of our knowledge, all previously investigated species produced orthodox seeds and no data on phosphoproteomic analysis of non-orthodox or recalcitrant seeds have been published.</p><p>The characterisation of the phosphoproteome includes the detection and identification of phosphoproteins and phosphopeptides, localisation of the exact phosphorylation sites and the quantitation of phosphorylation status, which can be performed by gel-based and gel-free approaches. Although several MS-based approaches for studying phosphoproteins, including down and bottom-up ones (Kaufmann et al., <xref rid="B22" ref-type="bibr">2001</xref>; Woods Ignatoski, <xref rid="B57" ref-type="bibr">2001</xref>; Agrawal and Thelen, <xref rid="B1" ref-type="bibr">2005</xref>) have been used, phosphopeptides are notoriously difficult to analyse, especially in the presence of their non-phosphorylated counterparts. This is due, among other factors, to the low stoichiometry of phosphorylated proteins arising from the fact that only a small fraction of the protein will exist in a particular phosphorylated form (Wu et al., <xref rid="B58" ref-type="bibr">2011</xref>; Rigbolt and Blagoev, <xref rid="B38" ref-type="bibr">2012</xref>).</p><p>Phosphoproteomic experiments are being perfomed by using a phospho-protein/peptide enrichment preliminary step (Thingholm et al., <xref rid="B49" ref-type="bibr">2009</xref>). These protocols require an excessive manipulation of the sample, thus reducing the confidence of the comparative results. It is for that reason, and as a complementary protocol, we propose the use of multiplexing (SYPRO-Ruby and Pro-Q DPS) staining of high-resolution 2-DE gels for a simultaneous analysis of protein changes in abundance and/or phosphorylation status.</p><p>In the present work we describe the use of that a technique to detect changes in the phosphoprotein profile throughout the <italic>Q. ilex</italic> seed germination and early seedling growth stages. After MALDI-TOF-TOF MS analysis, we have identified 20 proteins whose phosphorylation status varies during the seed developmental process, with seven of them showing no differences in abundance. This last group included enzymes of the glycolytic and amino acid pathways that were, respectively, more and less phosphorylated in seedlings than in seeds. This pattern was different from the one reported for orthodox seed species, in which concomitant changes in abundance and phosphorylation have been observed for enzymes of these two pathways.</p></sec><sec sec-type="materials|methods" id="s2"><title>Materials and methods</title><sec><title>Plant material</title><p>Mature acorns were harvested during October–November from healthy holm oaks from Cerro Muriano-Córdoba (Córdoba, Spain 37°59′57.74″N, 4°46′57.93″W). Germination and seedling growth were performed at 22 ± 1°C for up to 10 days in darkness as described in Liu et al. (<xref rid="B30" ref-type="bibr">2012</xref>). Undamaged, mature acorns were sterilized by immersion in 2.5% sodium hypochlorite, washed abundantly with water and finally dried with filter paper. In order to achive a homogeneous and synchronized germination (Liu et al., <xref rid="B30" ref-type="bibr">2012</xref>), acorns were peeled, removing the pericarp and cutting off parts of the distal ends of the acorns, and then placed in plastic boxes containing one sheet of whatman No3 filter paper over wet perlite. The system was covered with filter paper to avoid water loss (Figure <xref ref-type="supplementary-material" rid="SM2">S1</xref>). Analyzed time course/periods, corresponding to different seed developmental stages (Figure <xref ref-type="supplementary-material" rid="SM2">S2</xref>) was selected based on morphology as assessed by microscopic observations (Romero-Rodríguez, <xref rid="B39" ref-type="bibr">2015</xref>; Ph. D Thesis); these stages were selected because they were representative of the morphological changes that occur during germination and seedling growth (24 h after imbibition the emergence of radicle was visible and 216 h shoot seedling started to grow). The embryonic axis was removed from seeds at 0 and 24 h after imbibition and the whole seedling at 216 h after imbibition. Samples from each time were abundantly washed with water, blot dried and frozen in liquid nitrogen and stored at −80°C until protein extraction. Three pooled samples per stage, each one corresponding to a biological replicate (1–2 g fresh weight per pool coming from 20 to 100 individuals), were performed.</p></sec><sec><title>Protein extraction, 2-DE electrophoresis and multiplex staining of the gels</title><p>Tissue samples were ground to a fine powder in liquid nitrogen using a mortar and pestle (three biological replicates per stage). Protein extracts were obtained from embryo axes of mature (0 h, un-imbibed seeds) and germinated seeds (24 h after imbibition when the radicle just emerged) and from seedling radicles (4.5–5 cm length, 216 h after imbibition), (Figure <xref ref-type="supplementary-material" rid="SM2">S2</xref>). Proteins were extracted using TCA/acetone-phenol according to the protocol of Wang et al. (<xref rid="B56" ref-type="bibr">2006</xref>). Protein content in samples was estimated by the method of Bradford (Bradford, <xref rid="B8" ref-type="bibr">1976</xref>) with bovine serum albumin as a standard. Samples (400 μg of protein) of each biological replicate per gel, were focused on 17 cm, 5–8 pH IPG strips using a Bio–Rad Protean IEF Cell system (Görg et al., <xref rid="B16" ref-type="bibr">2004</xref>; Maldonado et al., <xref rid="B32" ref-type="bibr">2008</xref>; Valero Galván et al., <xref rid="B52" ref-type="bibr">2011</xref>). The second dimension, SDS-PAGE (Laemmli, <xref rid="B25" ref-type="bibr">1970</xref>) was performed on 12% polyacrylamide gels (PROTEAN® Plus Dodeca Cell). Gels were double stained, first with Pro-Q DPS and then with SYPRO-Ruby (Figure <xref ref-type="supplementary-material" rid="SM2">S3</xref>) following the procedure described in Agrawal and Thelen (<xref rid="B2" ref-type="bibr">2006</xref>) and Berggren et al. (<xref rid="B6" ref-type="bibr">2000</xref>). Images were captured with Molecular Imager FX (Bio-Rad Laboratories, Inc.). Experimental <italic>Mr</italic>-values were calculated by mobility comparisons with protein standard markers (SDS-PAGE Standards, 161-0304, Bio-Rad) run in a separate marker lane on the SDS-gel, while pI was determined by using a 5–8 linear scale over the total length of the IPG strips.</p></sec><sec><title>Gel image analysis and statistical tests</title><p>Gel image (Pro-Q DPS and SYPRO-Ruby) analysis was performed with PDQuest 8.0.1 software (Bio-Rad) (Valledor and Jorrín, <xref rid="B54" ref-type="bibr">2011</xref>). As reported by Agrawal and Thelen (<xref rid="B2" ref-type="bibr">2006</xref>) and in order to eliminate false positives, phosphoproteins spots (revealed with Pro-Q DPS) were only considered if the Pro-Q DPS/SYPRO-Ruby volume ratios were higher than those obtained for negative control, non-phosphorylated markers (β-galactosidase and serum albumin) and with ratios equal to or higher than those obtained for phosphorylated ovoalbumin used as positive control. Consistent spot volumes (those present in all biological replicates) were normalized based on total quantity in valid spots, calculated for each 2-DE gel and used for statistical assessments of differential phosphoprotein and total protein abundance. For statistical analysis (ANOVA, PCA), the web-based software NIA array analysis tool (<ext-link ext-link-type="uri" xlink:href="http://lgsun.grc.nia.nih.gov/anova/index.html">http://lgsun.grc.nia.nih.gov/anova/index.html</ext-link>) (Sharov et al., <xref rid="B44" ref-type="bibr">2005</xref>; Sghaier-Hammami et al., <xref rid="B43" ref-type="bibr">2013</xref>) was employed.</p></sec><sec><title>MALDI-TOF/TOF analysis</title><p>Spots with differential abundance were automatically excised (Investigator ProPic, Genomic Solutions), transferred to multiwell 96 plates, and digested with modified porcine trypsin (sequencing grade; Promega) by using a ProGest (Genomics Solution) digestion station. In-gel digestion was performed as decribed by Shevchenko et al. (<xref rid="B45" ref-type="bibr">1996</xref>). Peptides were extracted from gel plugs by adding 10 μL of 10% (v/v) trifluoracetic acid (15 min at room temperature). Solubilized peptides were desalted and concentrated by using μC-18 ZipTip columns (Millipore). Eluate was directly loaded onto the MALDI plate using α-cyano hydroxycinnamic acid as a matrix. Peptide mass analysis was performed with a MALDI-TOF/TOF (4800 Proteomics Analyzer, Applied Biosystems). The most abundant peptide ions were then subjected to fragmentation analysis (MS/MS), providing information that can be used to determine the peptide sequence. Proteins were assigned identification by peptide mass fingerprinting and confirmed by MS/MS analysis. Mascot 2.0 search engine (Matrix Science Ltd., London; <ext-link ext-link-type="uri" xlink:href="http://www.matrixscience.com">http://www.matrixscience.com</ext-link>) was used for protein identification running over non-redundant NCBI protein, UniprotKB, and <italic>Quercus</italic> (Romero-Rodriguez et al., <xref rid="B40" ref-type="bibr">2014</xref>) databases. The following parameters were allowed: taxonomy restrictions to <italic>Viridiplantae</italic> in public databases, one missed cleavage, 100 ppm mass tolerance in MS and 0.5 Da for MS/MS data, cysteine carbamidomethylation as a fixed modification, methionine oxidation, and the phosphorylation of Ser, Thr, and Tyr residues as a variable modification. The confidence in the peptide mass fingerprinting matches (<italic>p</italic> < 0.05) was based on the MOWSE score, and confirmed by the accurate overlapping of the matched peptides with the major peaks of the mass spectrum. Proteins with statistically significant (<italic>p</italic> < 0.05) hits were positively assigned identification. Identified phosphoprotein sequences downloaded from UniprotKB, NCBI nr or available in Quercus_DB (Romero-Rodriguez et al., <xref rid="B40" ref-type="bibr">2014</xref>) were subjected to BLAST analysis by using the phosphoprotein BLAST tool in the Plant Protein Phosphorylation DataBase (P3DB) (Gao et al., <xref rid="B15" ref-type="bibr">2009</xref>) available at <ext-link ext-link-type="uri" xlink:href="http://www.p3db.org/">http://www.p3db.org/</ext-link>, to find orthologous proteins whose phosphorylation sites were described previously in other species. Proteins identified by MALDI TOF/TOF analysis were extracted and classified based on their putative function according to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, using Blast2GO (Conesa et al., <xref rid="B10" ref-type="bibr">2005</xref>) based on BLASTp results against NCBI nr protein database (<italic>e</italic> < 10<sup>−3</sup>), or according to annotations in UniProtKB protein database.</p></sec></sec><sec sec-type="results" id="s3"><title>Results</title><p>By using a multiplex double staining of the gels it was possible to detect changes in the protein abundance (SYPRO-Ruby-stained spots) and phosphorylation status (Pro-Q DPS-stained spots) throughout the seed germination and early seedling growth of <italic>Q. ilex</italic> in three different stages, mature seeds (just before imbibition), germinated seeds (24 h after imbibition), and early grown seedlings (216 h after imbibition) (Figure <xref ref-type="supplementary-material" rid="SM2">S2</xref>). In the analyzed stages the protein yield per fresh weight was around 2–15 mg of protein per g, diminishing during the seedling growth (Table <xref ref-type="table" rid="T1">1</xref>).</p><table-wrap id="T1" position="float"><label>Table 1</label><caption><p><bold>Electrophoretic analysis of changes in the protein and phosphoprotein profile during germination and seedling growth</bold>.</p></caption><table frame="hsides" rules="groups"><thead><tr><th valign="top" align="left" rowspan="1" colspan="1"><bold>Hours after imbibition</bold></th><th valign="top" align="center" rowspan="1" colspan="1"><bold>Protein yield mg g<sup>−1</sup> of fresh weight</bold></th><th valign="top" align="center" rowspan="1" colspan="1"><bold>Spots detected by SYPRO-Ruby stain</bold></th><th valign="top" align="center" rowspan="1" colspan="1"><bold>Spots detected by Pro-Q DPS</bold></th><th valign="top" align="center" colspan="4" rowspan="1"><bold>Spots with change in total protein profile<xref ref-type="table-fn" rid="TN1"><sup>*</sup></xref> (SYPRO-Ruby)</bold></th><th valign="top" align="center" colspan="4" rowspan="1"><bold>Spots with change in phosphoprotein profile<xref ref-type="table-fn" rid="TN1"><sup>*</sup></xref></bold></th></tr><tr><th rowspan="1" colspan="1"></th><th rowspan="1" colspan="1"></th><th rowspan="1" colspan="1"></th><th rowspan="1" colspan="1"></th><th valign="top" align="center" colspan="2" rowspan="1"><bold>Qualitative</bold></th><th valign="top" align="center" colspan="2" rowspan="1"><bold>Quantitative</bold></th><th valign="top" align="center" colspan="2" rowspan="1"><bold>Qualitative</bold></th><th valign="top" align="center" colspan="2" rowspan="1"><bold>Quantitative</bold></th></tr><tr><th rowspan="1" colspan="1"></th><th rowspan="1" colspan="1"></th><th rowspan="1" colspan="1"></th><th rowspan="1" colspan="1"></th><th valign="top" align="center" rowspan="1" colspan="1"><bold>Up</bold></th><th valign="top" align="center" rowspan="1" colspan="1"><bold>Down</bold></th><th valign="top" align="center" rowspan="1" colspan="1"><bold>Up</bold></th><th valign="top" align="center" rowspan="1" colspan="1"><bold>Down</bold></th><th valign="top" align="center" rowspan="1" colspan="1"><bold>Up</bold></th><th valign="top" align="center" rowspan="1" colspan="1"><bold>Down</bold></th><th valign="top" align="center" rowspan="1" colspan="1"><bold>Up</bold></th><th valign="top" align="center" rowspan="1" colspan="1"><bold>Down</bold></th></tr></thead><tbody><tr><td valign="top" align="left" rowspan="1" colspan="1">0</td><td valign="top" align="center" rowspan="1" colspan="1">15.2</td><td valign="top" align="center" rowspan="1" colspan="1">402</td><td valign="top" align="center" rowspan="1" colspan="1">205</td><td valign="top" align="center" rowspan="1" colspan="1">–</td><td valign="top" align="center" rowspan="1" colspan="1">–</td><td valign="top" align="center" rowspan="1" colspan="1">–</td><td valign="top" align="center" rowspan="1" colspan="1">–</td><td valign="top" align="center" rowspan="1" colspan="1">–</td><td valign="top" align="center" rowspan="1" colspan="1">–</td><td valign="top" align="center" rowspan="1" colspan="1">–</td><td valign="top" align="center" rowspan="1" colspan="1">–</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">24</td><td valign="top" align="center" rowspan="1" colspan="1">13.6</td><td valign="top" align="center" rowspan="1" colspan="1">412</td><td valign="top" align="center" rowspan="1" colspan="1">211</td><td valign="top" align="center" rowspan="1" colspan="1">3</td><td valign="top" align="center" rowspan="1" colspan="1">3</td><td valign="top" align="center" rowspan="1" colspan="1">2</td><td valign="top" align="center" rowspan="1" colspan="1">3</td><td valign="top" align="center" rowspan="1" colspan="1">1</td><td valign="top" align="center" rowspan="1" colspan="1">6</td><td valign="top" align="center" rowspan="1" colspan="1">4</td><td valign="top" align="center" rowspan="1" colspan="1">2</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">216</td><td valign="top" align="center" rowspan="1" colspan="1">1.9</td><td valign="top" align="center" rowspan="1" colspan="1">329</td><td valign="top" align="center" rowspan="1" colspan="1">174</td><td valign="top" align="center" rowspan="1" colspan="1">17</td><td valign="top" align="center" rowspan="1" colspan="1">8</td><td valign="top" align="center" rowspan="1" colspan="1">33</td><td valign="top" align="center" rowspan="1" colspan="1">23</td><td valign="top" align="center" rowspan="1" colspan="1">7</td><td valign="top" align="center" rowspan="1" colspan="1">26</td><td valign="top" align="center" rowspan="1" colspan="1">12</td><td valign="top" align="center" rowspan="1" colspan="1">8</td></tr></tbody></table><table-wrap-foot><p><italic>Up and down accumulated proteins were calculated respect to the mature (0h) stage</italic>.</p><fn id="TN1"><label>*</label><p><italic>Phosphorylation profile was considered changed when no difference was observed in SYPRO-Ruby staining but was statistically different in Pro-Q DPS. In contrast, it was considered unchanged when a difference was observed in both staining methods</italic>.</p></fn><p><italic>Number of spots detected by SYPRO-Ruby and Pro-Q DPS in different analyzed stages and number of differential spots in total protein and phosphoprotein are shown</italic>.</p></table-wrap-foot></table-wrap><p>Proteins in the extract were separated by 2-DE, and were evenly distributed throughout along the whole pH (5–8) and <italic>Mr</italic> (6–116 kDa) ranges (Figure <xref ref-type="fig" rid="F1">1</xref>). A total of 482 spots were resolved after SYPRO-Ruby staining, with 222 of them also being stained with Pro-Q DPS, these corresponding to putative phosphoproteins (Table <xref ref-type="supplementary-material" rid="SM1">S1</xref>).</p><fig id="F1" position="float"><label>Figure 1</label><caption><p><bold>A virtual 2-DE gel showing the protein profile of <italic><bold>Q. ilex</bold></italic> mature seed embryo axis (0 h, un-imbibed) obtained by successive Pro-Q DPS and SYPRO-Ruby staining</bold>. Proteins stained with SYPRO-Ruby appear in green, while Pro-Q DPS stained proteins appear in red. The statistically significant differential phosphoprotein spots are indicated with circles for quantitative differences and with triangles for qualitative (absence/presence) differences. Numbers in red indicate the protein spots that were identified by MALDI TOF/TOF.</p></caption><graphic xlink:href="fpls-06-00620-g0001"></graphic></fig><p>Consistent Pro-Q DPS stained spots, present in all the three biological replicates, were subjected to statistical, ANOVA and PCA, analysis, with 55, out of the 222, showing significant variations (spot volume) between samples (Table <xref ref-type="supplementary-material" rid="SM1">S1</xref>). Both qualitative and quantitative changes were observed (Table <xref ref-type="table" rid="T1">1</xref>). Taking as a reference the mature seed phosphoprotein-profile, big changes occur after radicle emergence, with small differences in germinated seeds. At the seedling stage (216 h post imbibition), 33 qualitative (7 newly appeared and 26 disappeared), and 20 quantitative (12 up and 8 down) changes were observed (Table <xref ref-type="table" rid="T1">1</xref>).</p><p>Two-dimensional biplots indicating associations between experimental samples and protein spots were generated by principal component analysis (PCA) in NIA array analysis tools (Figure <xref ref-type="fig" rid="F2">2</xref>). The consistent Pro-Q-DPS stained spots were different enough to establish groups of the samples analyzed. The three analyzed stages were separated from each other; the first component separated the mature (0 h, un-imbibed) and germinated (24 h after imbibition) stage from seedling stages (216 h post imbibition), and the second component separated all the three stages. PCA results showed that PC1 and PC2 explained 88.57 and 11.42% of total variance, respectively. The 55 putative phosphoprotein spots were selected for MALDI-TOF/TOF MS analysis. ANOVA tests of SYPRO-Ruby stained spots (Table <xref ref-type="supplementary-material" rid="SM1">S1</xref> and Figure <xref ref-type="supplementary-material" rid="SM2">S4</xref>) revealed that 20 out of the 55 did not show any differences in abundance while 35 did.</p><fig id="F2" position="float"><label>Figure 2</label><caption><p><bold>Principal component analysis plots. (A)</bold> Representation of the samples based on the main principal components found after PCA. <bold>(B)</bold> Plot component PC1 vs. PC2 of differentially expressed spots in three stages analyzed.</p></caption><graphic xlink:href="fpls-06-00620-g0002"></graphic></fig><sec><title>Protein identification</title><p>After MALDI-TOF/TOF analysis, 20 putative phosphoproteins were identified (Table <xref ref-type="table" rid="T2">2</xref>). Out of the 20, 13 changed in abundance, while seven did not. For the former the variation in phospho-signal could be simply due to a change in abundance while for the other seven results can only be explained by a modification in their phosphorylation status. To validate the phosphoprotein character/nature of the identified proteins, a BLAST against entries at Plant Protein Phosphorylation DataBase (P3DB; <ext-link ext-link-type="uri" xlink:href="http://www.p3db.org/">http://www.p3db.org/</ext-link>) (Kersten et al., <xref rid="B23" ref-type="bibr">2009</xref>) was performed. With the only exception of spot 3103, a putative cyclase family protein, the other <italic>Q. ilex</italic> proteins had at least one orthologous phosphoprotein in <italic>A. thaliana, M. truncatula, Nicotiana tabacum</italic>, and <italic>Glycine max</italic>. Table <xref ref-type="table" rid="T3">3</xref> lists the orthologous proteins and their host species (Sugiyama et al., <xref rid="B47" ref-type="bibr">2008</xref>; Jones et al., <xref rid="B20" ref-type="bibr">2009</xref>; Li et al., <xref rid="B27" ref-type="bibr">2009</xref>; Reiland et al., <xref rid="B37" ref-type="bibr">2009</xref>; Grimsrud et al., <xref rid="B17" ref-type="bibr">2010</xref>; Nakagami et al., <xref rid="B35" ref-type="bibr">2010</xref>; Fíla et al., <xref rid="B12" ref-type="bibr">2012</xref>; Rose et al., <xref rid="B42" ref-type="bibr">2012b</xref>).</p><table-wrap id="T2" position="float"><label>Table 2</label><caption><p><bold>List of proteins identified by MALDI TOF/TOF, grouped in functional categories, based on the KEGG pathways database</bold>.</p></caption><table frame="hsides" rules="groups"><thead><tr><th valign="top" align="left" rowspan="1" colspan="1"><bold>Numbers<xref ref-type="table-fn" rid="TN1a"><sup>a</sup></xref></bold></th><th valign="top" align="left" rowspan="1" colspan="1"><bold>Protein name</bold></th><th valign="top" align="left" rowspan="1" colspan="1"><bold>Accession numbers<xref ref-type="table-fn" rid="TN2a"><sup>b</sup></xref></bold></th><th valign="top" align="center" colspan="2" rowspan="1"><bold>Mr (pI)</bold></th><th valign="top" align="center" rowspan="1" colspan="1"><bold>MOWSE score<xref ref-type="table-fn" rid="TN5a"><sup>e</sup></xref></bold></th><th valign="top" align="center" rowspan="1" colspan="1"><bold>Peptide matches</bold></th><th valign="top" align="center" rowspan="1" colspan="1"><bold>Seq cov (%)</bold></th><th valign="top" align="left" rowspan="1" colspan="1"><bold>Fragmented ion (Ion Score)</bold></th><th valign="top" align="center" rowspan="1" colspan="1"><bold>Normalized spot volume<xref ref-type="table-fn" rid="TN6a"><sup>f</sup></xref></bold></th></tr><tr><th rowspan="1" colspan="1"></th><th rowspan="1" colspan="1"></th><th rowspan="1" colspan="1"></th><th valign="top" align="left" rowspan="1" colspan="1"><bold>Theor<xref ref-type="table-fn" rid="TN3a"><sup>c</sup></xref></bold></th><th valign="top" align="left" rowspan="1" colspan="1"><bold>Exp<xref ref-type="table-fn" rid="TN4a"><sup>d</sup></xref></bold></th><th rowspan="1" colspan="1"></th><th rowspan="1" colspan="1"></th><th rowspan="1" colspan="1"></th><th rowspan="1" colspan="1"></th><th rowspan="1" colspan="1"></th></tr></thead><tbody><tr><td valign="top" align="left" colspan="10" style="background-color:#bbbdc0" rowspan="1"><bold>PHOSPHOPROTEIN SPOTS IN 2-DE WITH CHANGES IN PHOSPHOPROTEIN STATUS</bold></td></tr><tr><td valign="top" align="left" colspan="10" rowspan="1"><bold>Carbohydrate Metabolism</bold></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">7502</td><td valign="top" align="left" rowspan="1" colspan="1">Pyrophosphate-dependent phosphofructokinase beta subunit. <italic>Citrus sinensis</italic> x <italic>Citrus trifoliate</italic></td><td valign="top" align="left" rowspan="1" colspan="1"><ext-link ext-link-type="DDBJ/EMBL/GenBank" xlink:href="A9YVC9">A9YVC9</ext-link></td><td valign="top" align="left" rowspan="1" colspan="1">62.0 (6.3)</td><td valign="top" align="left" rowspan="1" colspan="1">53.8 (6.8)</td><td valign="top" align="center" rowspan="1" colspan="1">283</td><td valign="top" align="center" rowspan="1" colspan="1">17</td><td valign="top" align="center" rowspan="1" colspan="1">22</td><td valign="top" align="left" rowspan="1" colspan="1">DKIETPEQFK (59)<break></break>STGKYYHFVR (45)<break></break>YYHFVR (37)<break></break>GQSHFFGYEGR (83)</td><td valign="top" align="left" rowspan="1" colspan="1"><inline-graphic xlink:href="fpls-06-00620-i0001.jpg"></inline-graphic></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">7318</td><td valign="top" align="left" rowspan="1" colspan="1">Phosphoglycerate kinase_AT1G79550.1, <italic>Quercus rubra</italic></td><td valign="top" align="left" rowspan="1" colspan="1"><ext-link ext-link-type="DDBJ/EMBL/GenBank" xlink:href="QRU405_58">QRU405_58</ext-link></td><td valign="top" align="left" rowspan="1" colspan="1">43.5 (6.7)</td><td valign="top" align="left" rowspan="1" colspan="1">40.0 (7.1)</td><td valign="top" align="center" rowspan="1" colspan="1">231</td><td valign="top" align="center" rowspan="1" colspan="1">11</td><td valign="top" align="center" rowspan="1" colspan="1">51</td><td valign="top" align="left" rowspan="1" colspan="1">YSLKPIVPR (31)<break></break>VILSTHLGRPK(30)<break></break>FLKPAVAGFLMQK(21)<break></break>LVAEIPEGGVLLLENVR(26)<break></break>LASLADLYVNDAFGTAHR (49)</td><td valign="top" align="left" rowspan="1" colspan="1"><inline-graphic xlink:href="fpls-06-00620-i0002.jpg"></inline-graphic></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">4304</td><td valign="top" align="left" rowspan="1" colspan="1">Glucose-1-phosphate adenylyltransferase, <italic>Vitis vinífera</italic></td><td valign="top" align="left" rowspan="1" colspan="1"><ext-link ext-link-type="DDBJ/EMBL/GenBank" xlink:href="D7TDB6">D7TDB6</ext-link></td><td valign="top" align="left" rowspan="1" colspan="1">56.2 (6.5)</td><td valign="top" align="left" rowspan="1" colspan="1">37.4 (6.1)</td><td valign="top" align="center" rowspan="1" colspan="1">350</td><td valign="top" align="center" rowspan="1" colspan="1">26</td><td valign="top" align="center" rowspan="1" colspan="1">38</td><td valign="top" align="left" rowspan="1" colspan="1">VDTTILGLDDER (59)<break></break>KPVPDFSFYDR (71)<break></break>SSPIYTQPR (41)<break></break>IINSDNVQEAAR (40)</td><td valign="top" align="left" rowspan="1" colspan="1"><inline-graphic xlink:href="fpls-06-00620-i0003.jpg"></inline-graphic></td></tr><tr><td valign="top" align="left" colspan="10" rowspan="1"><bold>Amino Acid Metabolism</bold></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">3612</td><td valign="top" align="left" rowspan="1" colspan="1">Glutamate decarboxylase_AT2G02010.1, <italic>Quercus</italic> spp.</td><td valign="top" align="left" rowspan="1" colspan="1"><ext-link ext-link-type="DDBJ/EMBL/GenBank" xlink:href="TC19169_41">TC19169_41</ext-link></td><td valign="top" align="left" rowspan="1" colspan="1">58.0 (5.9)</td><td valign="top" align="left" rowspan="1" colspan="1">50.2 (6.1)</td><td valign="top" align="center" rowspan="1" colspan="1">208</td><td valign="top" align="center" rowspan="1" colspan="1">12</td><td valign="top" align="center" rowspan="1" colspan="1">45</td><td valign="top" align="left" rowspan="1" colspan="1">VVIREDFSR (30)<break></break>ETPEEIATYWR (53)<break></break>GSSQIIAQYYQFVR (69)<break></break>NYVDMDEYPVTTELQNR (46)</td><td valign="top" align="left" rowspan="1" colspan="1"><inline-graphic xlink:href="fpls-06-00620-i0004.jpg"></inline-graphic></td></tr><tr><td valign="top" align="left" colspan="10" rowspan="1"><bold>Protein Folding</bold></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">2606</td><td valign="top" align="left" rowspan="1" colspan="1">Heat shock protein 60_AT3G23990.1, <italic>Quercus</italic> spp.</td><td valign="top" align="left" rowspan="1" colspan="1"><ext-link ext-link-type="DDBJ/EMBL/GenBank" xlink:href="TC33448_39">TC33448_39</ext-link></td><td valign="top" align="left" rowspan="1" colspan="1">63.9 (5.6)</td><td valign="top" align="left" rowspan="1" colspan="1">68.1 (5.8)</td><td valign="top" align="center" rowspan="1" colspan="1">436</td><td valign="top" align="center" rowspan="1" colspan="1">11</td><td valign="top" align="center" rowspan="1" colspan="1">23</td><td valign="top" align="left" rowspan="1" colspan="1">AGIIDPVKVIR (51)<break></break>IGVQIIQNALK (91)<break></break>NVVIEQSWGAPK (82)<break></break>GYISPYFITNQK (73)<break></break>SDEIAQVGTISANGER (72)<break></break>AAVEEGIVPGGGVALLYASK (75)</td><td valign="top" align="left" rowspan="1" colspan="1"><inline-graphic xlink:href="fpls-06-00620-i0005.jpg"></inline-graphic></td></tr><tr><td valign="top" align="left" colspan="10" rowspan="1"><bold>Unknown</bold></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">4107</td><td valign="top" align="left" rowspan="1" colspan="1">Unknown protein</td><td valign="top" align="left" rowspan="1" colspan="1"><ext-link ext-link-type="DDBJ/EMBL/GenBank" xlink:href="A9PFJ3">A9PFJ3</ext-link></td><td valign="top" align="left" rowspan="1" colspan="1">29.5 (6.2)</td><td valign="top" align="left" rowspan="1" colspan="1">20.2 (6.3)</td><td valign="top" align="center" rowspan="1" colspan="1">169</td><td valign="top" align="center" rowspan="1" colspan="1">9</td><td valign="top" align="center" rowspan="1" colspan="1">35</td><td valign="top" align="left" rowspan="1" colspan="1">LQGNYYFQEQLSR (95)<break></break>GSSIWYGCVLR (29)</td><td valign="top" align="left" rowspan="1" colspan="1"><inline-graphic xlink:href="fpls-06-00620-i0006.jpg"></inline-graphic></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">1711</td><td valign="top" align="left" rowspan="1" colspan="1">Cell division protein ftsH, putative, <italic>Ricinus communis</italic></td><td valign="top" align="left" rowspan="1" colspan="1"><ext-link ext-link-type="DDBJ/EMBL/GenBank" xlink:href="B9S304">B9S304</ext-link></td><td valign="top" align="left" rowspan="1" colspan="1">75.5 (6.4)</td><td valign="top" align="left" rowspan="1" colspan="1">78.7 (5.7)</td><td valign="top" align="center" rowspan="1" colspan="1">224</td><td valign="top" align="center" rowspan="1" colspan="1">21</td><td valign="top" align="center" rowspan="1" colspan="1">26</td><td valign="top" align="left" rowspan="1" colspan="1">FLEYLDKDR (48)<break></break>VRVQLPGLSQELLQK (3)<break></break>SSGGMGGPGGPGFPLAFGQSK (53)<break></break>ADILDSALLRPGR (17)</td><td valign="top" align="left" rowspan="1" colspan="1"><inline-graphic xlink:href="fpls-06-00620-i0007.jpg"></inline-graphic></td></tr><tr><td valign="top" align="left" colspan="10" style="background-color:#bbbdc0" rowspan="1"><bold>PHOSPHOPROTEIN WITHOUT CHANGES IN PHOSPHORYLATIONS STATUS</bold></td></tr><tr><td valign="top" align="left" colspan="10" rowspan="1"><bold>Carbohydrate Metabolism</bold></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">4610</td><td valign="top" align="left" rowspan="1" colspan="1">Pyruvate decarboxylase (<italic>Prunus armeniaca</italic>)</td><td valign="top" align="left" rowspan="1" colspan="1"><ext-link ext-link-type="DDBJ/EMBL/GenBank" xlink:href="B0ZS79">B0ZS79</ext-link></td><td valign="top" align="left" rowspan="1" colspan="1">66.2 (5.7)</td><td valign="top" align="left" rowspan="1" colspan="1">61.9 (6.2)</td><td valign="top" align="center" rowspan="1" colspan="1">235</td><td valign="top" align="center" rowspan="1" colspan="1">12</td><td valign="top" align="center" rowspan="1" colspan="1">23</td><td valign="top" align="left" rowspan="1" colspan="1">ILHHTIGLPDFSQELR(124)<break></break>EPVPFSLSPR(69)</td><td valign="top" align="left" rowspan="1" colspan="1"><inline-graphic xlink:href="fpls-06-00620-i0008.jpg"></inline-graphic></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">3609</td><td valign="top" align="left" rowspan="1" colspan="1">Phosphoglycerate mutase_AT1G09780.1 (<italic>Quercus petraea)</italic></td><td valign="top" align="left" rowspan="1" colspan="1"><ext-link ext-link-type="DDBJ/EMBL/GenBank" xlink:href="QP1063_77">QP1063_77</ext-link></td><td valign="top" align="left" rowspan="1" colspan="1">61.0 (6.0)</td><td valign="top" align="left" rowspan="1" colspan="1">60.5 (6.0)</td><td valign="top" align="center" rowspan="1" colspan="1">476</td><td valign="top" align="center" rowspan="1" colspan="1">10</td><td valign="top" align="center" rowspan="1" colspan="1">25</td><td valign="top" align="left" rowspan="1" colspan="1">DAILSGKFDQVR (46)<break></break>FGHVTFFWNGNR (77)<break></break>AFEYEDFDKFDR (67)<break></break>LPSHYLVSPPEIDR (78)<break></break>GTLHLIGLLSDGGVHSR (98)<break></break>IQILTSHTCQPVPIAIGGPGLAPGCR (88)<break></break>AHGSAVGLPTEDDMGNSEVGHNALGAGR (31)</td><td valign="top" align="left" rowspan="1" colspan="1"><inline-graphic xlink:href="fpls-06-00620-i0009.jpg"></inline-graphic></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">5604</td><td valign="top" align="left" rowspan="1" colspan="1">Pyruvate decarboxylase, putative (<italic>Ricinus communis)</italic></td><td valign="top" align="left" rowspan="1" colspan="1"><ext-link ext-link-type="DDBJ/EMBL/GenBank" xlink:href="255563082">255563082</ext-link></td><td valign="top" align="left" rowspan="1" colspan="1">64.2 (5.9)</td><td valign="top" align="left" rowspan="1" colspan="1">61.1 (6.3)</td><td valign="top" align="center" rowspan="1" colspan="1">112</td><td valign="top" align="center" rowspan="1" colspan="1">2</td><td valign="top" align="center" rowspan="1" colspan="1">4</td><td valign="top" align="left" rowspan="1" colspan="1">ILHHTIGLPDFSQELR (97)<break></break>IFVP<italic><bold>S</bold></italic>GVPLK (22)<break></break>Phosphorilated S379<xref ref-type="table-fn" rid="TN7a"><sup>g</sup></xref></td><td valign="top" align="left" rowspan="1" colspan="1"><inline-graphic xlink:href="fpls-06-00620-i0010.jpg"></inline-graphic></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">3507</td><td valign="top" align="left" rowspan="1" colspan="1">Beta glucosidase 17_AT2G44480.1 (<italic>Quercus</italic> spp.)</td><td valign="top" align="left" rowspan="1" colspan="1"><ext-link ext-link-type="DDBJ/EMBL/GenBank" xlink:href="QRO15180_40">QRO15180_40</ext-link></td><td valign="top" align="left" rowspan="1" colspan="1">47.8 (5.2)</td><td valign="top" align="left" rowspan="1" colspan="1">60.3 (6.2)</td><td valign="top" align="center" rowspan="1" colspan="1">119</td><td valign="top" align="center" rowspan="1" colspan="1">10</td><td valign="top" align="center" rowspan="1" colspan="1">48</td><td valign="top" align="left" rowspan="1" colspan="1">GAYDFIGVNYYTSR (103)</td><td valign="top" align="left" rowspan="1" colspan="1"><inline-graphic xlink:href="fpls-06-00620-i0011.jpg"></inline-graphic></td></tr><tr><td valign="top" align="left" colspan="10" rowspan="1"><bold>Amino Acid Metabolism</bold></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">7702</td><td valign="top" align="left" rowspan="1" colspan="1">5-methyltetrahydropteroyltriglutamate–homocysteine methyltransferase-like (<italic>Solanum lycopersicum)</italic></td><td valign="top" align="left" rowspan="1" colspan="1"><ext-link ext-link-type="DDBJ/EMBL/GenBank" xlink:href="460407874">460407874</ext-link></td><td valign="top" align="left" rowspan="1" colspan="1">85.0 (6.0)</td><td valign="top" align="left" rowspan="1" colspan="1">82.5 (7.0)</td><td valign="top" align="center" rowspan="1" colspan="1">304</td><td valign="top" align="center" rowspan="1" colspan="1">4</td><td valign="top" align="center" rowspan="1" colspan="1">6</td><td valign="top" align="left" rowspan="1" colspan="1">YLFAGVVDGR(76)<break></break>ALSGAKDEAFF<italic><bold>S</bold></italic>ANAAAQASR(41)<break></break>Phosphorilated S378<xref ref-type="table-fn" rid="TN7a"><sup>g</sup></xref>EGVKYGAGIGPGVYDIHSPR (64)<break></break>YGAGIGPGVYDIHSPR(122)</td><td valign="top" align="left" rowspan="1" colspan="1"><inline-graphic xlink:href="fpls-06-00620-i0012.jpg"></inline-graphic></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">4303</td><td valign="top" align="left" rowspan="1" colspan="1">S-adenosylmethionine synthase 2 (<italic>Elaeagnus umbellate</italic>)</td><td valign="top" align="left" rowspan="1" colspan="1"><ext-link ext-link-type="DDBJ/EMBL/GenBank" xlink:href="Q9AT55">Q9AT55</ext-link></td><td valign="top" align="left" rowspan="1" colspan="1">43.6 (5.5)</td><td valign="top" align="left" rowspan="1" colspan="1">47.0 (6.2)</td><td valign="top" align="center" rowspan="1" colspan="1">650</td><td valign="top" align="center" rowspan="1" colspan="1">22</td><td valign="top" align="center" rowspan="1" colspan="1">44</td><td valign="top" align="left" rowspan="1" colspan="1">TIGFVSDDVGLDADNCK (83)<break></break>VLVNIEQQSPDIAQGVHGHFTK(97)<break></break>TQVTVEYYNDKGAMVPVR(17)<break></break>TIFHLNPSGR(61)<break></break>FVIGGPHGDAGLTGR(110)<break></break>FVIGGPHGDAGLTGRK (89)<break></break>TAAYGHFGR (79)</td><td valign="top" align="left" rowspan="1" colspan="1"><inline-graphic xlink:href="fpls-06-00620-i0013.jpg"></inline-graphic></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">3103</td><td valign="top" align="left" rowspan="1" colspan="1">Putative cyclase family protein (<italic>Arachis hypogaea</italic>)</td><td valign="top" align="left" rowspan="1" colspan="1"><ext-link ext-link-type="DDBJ/EMBL/GenBank" xlink:href="C0L2U1">C0L2U1</ext-link></td><td valign="top" align="left" rowspan="1" colspan="1">31.5 (5.04)</td><td valign="top" align="left" rowspan="1" colspan="1">30.7 (6.5)</td><td valign="top" align="center" rowspan="1" colspan="1">79</td><td valign="top" align="center" rowspan="1" colspan="1">8</td><td valign="top" align="center" rowspan="1" colspan="1">28</td><td valign="top" align="left" rowspan="1" colspan="1">IFDISHR (36)</td><td valign="top" align="left" rowspan="1" colspan="1"><inline-graphic xlink:href="fpls-06-00620-i0014.jpg"></inline-graphic></td></tr><tr><td valign="top" align="left" colspan="10" rowspan="1"><bold>Oxidation-reduction Process</bold></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">4103</td><td valign="top" align="left" rowspan="1" colspan="1">Glutathione S-transferase omega_D6BR66 (<italic>Quercus</italic> spp.)</td><td valign="top" align="left" rowspan="1" colspan="1"><ext-link ext-link-type="DDBJ/EMBL/GenBank" xlink:href="TC18312_19">TC18312_19</ext-link></td><td valign="top" align="left" rowspan="1" colspan="1">28.2 (6.6)</td><td valign="top" align="left" rowspan="1" colspan="1">26.0 (6.0)</td><td valign="top" align="center" rowspan="1" colspan="1">75</td><td valign="top" align="center" rowspan="1" colspan="1">8</td><td valign="top" align="center" rowspan="1" colspan="1">36</td><td valign="top" align="left" rowspan="1" colspan="1">LYISLSCPYAQR (24)<break></break>EAGPAFDHLENALSK (14)<break></break>WIEEVNKIDAYKPTK (11)<break></break>YIDSNFEGPSLLPNDHAK (24)</td><td valign="top" align="left" rowspan="1" colspan="1"><inline-graphic xlink:href="fpls-06-00620-i0015.jpg"></inline-graphic></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">5003</td><td valign="top" align="left" rowspan="1" colspan="1">Putative uncharacterized protein (Glutathione-s-transferase theta_B9T0U8) (<italic>Vitis vinífera</italic>)</td><td valign="top" align="left" rowspan="1" colspan="1"><ext-link ext-link-type="DDBJ/EMBL/GenBank" xlink:href="D7TP00">D7TP00</ext-link></td><td valign="top" align="left" rowspan="1" colspan="1">24.9 (6.2)</td><td valign="top" align="left" rowspan="1" colspan="1">15.3(6.6)</td><td valign="top" align="center" rowspan="1" colspan="1">107</td><td valign="top" align="center" rowspan="1" colspan="1">6</td><td valign="top" align="center" rowspan="1" colspan="1">22</td><td valign="top" align="left" rowspan="1" colspan="1">NPFGQIPVLEDGDLTLFESR (38)<break></break>AWWEDISSRPAFK (46)</td><td valign="top" align="left" rowspan="1" colspan="1"><inline-graphic xlink:href="fpls-06-00620-i0016.jpg"></inline-graphic></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">6003</td><td valign="top" align="left" rowspan="1" colspan="1">Manganese superoxide dismutase 1_AT3G10920.1 (<italic>Quercus</italic> spp.)</td><td valign="top" align="left" rowspan="1" colspan="1"><ext-link ext-link-type="DDBJ/EMBL/GenBank" xlink:href="TC29211_11">TC29211_11</ext-link></td><td valign="top" align="left" rowspan="1" colspan="1">19.3 (7.9)</td><td valign="top" align="left" rowspan="1" colspan="1">15.4 (6.8)</td><td valign="top" align="center" rowspan="1" colspan="1">126</td><td valign="top" align="center" rowspan="1" colspan="1">5</td><td valign="top" align="center" rowspan="1" colspan="1">55</td><td valign="top" align="left" rowspan="1" colspan="1">HHQAYITNYNK (73)<break></break>FNGGGHINHSIFWK (42)<break></break>KLVVDTTANQDPLVTK(2)</td><td valign="top" align="left" rowspan="1" colspan="1"><inline-graphic xlink:href="fpls-06-00620-i0017.jpg"></inline-graphic></td></tr><tr><td valign="top" align="left" colspan="10" rowspan="1"><bold>RNA Metabolism</bold></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">1303</td><td valign="top" align="left" rowspan="1" colspan="1">DEAD box RNA helicase (<italic>Pisum sativum</italic>)</td><td valign="top" align="left" rowspan="1" colspan="1"><ext-link ext-link-type="DDBJ/EMBL/GenBank" xlink:href="Q8H1A5">Q8H1A5</ext-link></td><td valign="top" align="left" rowspan="1" colspan="1">47.1 (5.4)</td><td valign="top" align="left" rowspan="1" colspan="1">49.6 (5.6)</td><td valign="top" align="center" rowspan="1" colspan="1">258</td><td valign="top" align="center" rowspan="1" colspan="1">24</td><td valign="top" align="center" rowspan="1" colspan="1">46</td><td valign="top" align="left" rowspan="1" colspan="1">GIYAYGFEKPSAIQQR (60)<break></break>ILSSGVHVVVGTPGR (28)<break></break>VFDMLRR (16)<break></break>MFVLDEADEMLSR (11)<break></break>VLITTDLLAR (21)</td><td valign="top" align="left" rowspan="1" colspan="1"><inline-graphic xlink:href="fpls-06-00620-i0018.jpg"></inline-graphic></td></tr><tr><td valign="top" align="left" colspan="10" rowspan="1"><bold>Stress Response</bold></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">4108</td><td valign="top" align="left" rowspan="1" colspan="1">Aluminum induced protein with YGL and LRDR motifs_AT3G22850.1 (<italic>Quercus</italic> spp.)</td><td valign="top" align="left" rowspan="1" colspan="1"><ext-link ext-link-type="DDBJ/EMBL/GenBank" xlink:href="TC18137_21">TC18137_21</ext-link></td><td valign="top" align="left" rowspan="1" colspan="1">27.8 (7.0)</td><td valign="top" align="left" rowspan="1" colspan="1">20.7 (6.3)</td><td valign="top" align="center" rowspan="1" colspan="1">141</td><td valign="top" align="center" rowspan="1" colspan="1">5</td><td valign="top" align="center" rowspan="1" colspan="1">27</td><td valign="top" align="left" rowspan="1" colspan="1">GCFFTSSGGLR(31)<break></break>FAFILYDSSSK (49)<break></break>SYEHPLNEVKPVPR (58)<break></break>SPEALQSPQSGSVSTLK (4)</td><td valign="top" align="left" rowspan="1" colspan="1"><inline-graphic xlink:href="fpls-06-00620-i0019.jpg"></inline-graphic></td></tr><tr><td valign="top" align="left" colspan="10" rowspan="1"><bold>Unknown</bold></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">8106</td><td valign="top" align="left" rowspan="1" colspan="1">AT4G39230.1_ NmrA-like negative transcriptional regulator family protein) (<italic>Quercus robur</italic>)</td><td valign="top" align="left" rowspan="1" colspan="1"><ext-link ext-link-type="DDBJ/EMBL/GenBank" xlink:href="QRO2324_17">QRO2324_17</ext-link></td><td valign="top" align="left" rowspan="1" colspan="1">36.5 (6.8)</td><td valign="top" align="left" rowspan="1" colspan="1">26.2 (7.6)</td><td valign="top" align="center" rowspan="1" colspan="1">367</td><td valign="top" align="center" rowspan="1" colspan="1">11</td><td valign="top" align="center" rowspan="1" colspan="1">35</td><td valign="top" align="left" rowspan="1" colspan="1">AGHPTFALVR(67)<break></break>VLIIGGTGYIGK(68)<break></break>FYPSEFGNDVDR(67)<break></break>AIFNKEDDIGTYTIK(17)<break></break>NLGVTLVHGDLYDHGSLVK(103<break></break>FYPSEFGNDVDRVHAVDPAK(17)<break></break>GDHTNFEIEPSFGVEASQLYPDVK(33)</td><td valign="top" align="left" rowspan="1" colspan="1"><inline-graphic xlink:href="fpls-06-00620-i0020.jpg"></inline-graphic></td></tr></tbody></table><table-wrap-foot><fn id="TN1a"><label>a</label><p><italic>Spot number as given on the 2-DE gel images in Figure <xref ref-type="fig" rid="F1">1</xref></italic>.</p></fn><fn id="TN2a"><label>b</label><p><italic>Uniprot, NCBI nr and Quercus_DB accessions numbers. The accession whose first letters were TC, QRU, QRO, and QP correspond to Quercus_DB. The accession numbers without letters correspond to NCBI nr</italic>.</p></fn><fn id="TN3a"><label>c</label><p><italic>Molecular weight (KDa) and isoelectric point of each database</italic>.</p></fn><fn id="TN4a"><label>d</label><p><italic>Molecular weight (kDa) and isoelectric point calculated by using molecular weight standards and the PD-Quest Advance (8.01) software</italic>.</p></fn><fn id="TN5a"><label>e</label><p><italic>Mascot score [S = −10 × log(P)]:where P is the probability that the observed match is a random event, peptide matched in MS analysis, percentage of sequence coverage, and ions sequence matched from MS-MS analysis</italic>.</p></fn><fn id="TN6a"><label>f</label><p><italic>The bar charts represent the normalized spot volume vs. analyzed stages</italic>.</p></fn><fn id="TN7a"><label>g</label><p><italic>The phosphorylation sites refer to the position of amino acids in the proteins</italic>.</p></fn></table-wrap-foot></table-wrap><table-wrap id="T3" position="float"><label>Table 3</label><caption><p><bold>Phosphorylation sites and close species in which the phosphoprotein identified in <italic><bold>Q. ilex</bold></italic> germinated seeds and seedling development was identified</bold>.</p></caption><table frame="hsides" rules="groups"><thead><tr><th valign="top" align="left" rowspan="1" colspan="1"><bold>Spot number</bold></th><th valign="top" align="left" rowspan="1" colspan="1"><bold>Protein name</bold></th><th valign="top" align="left" rowspan="1" colspan="1"><bold>Protein ID</bold></th><th valign="top" align="left" rowspan="1" colspan="1"><bold>Close species</bold></th><th valign="top" align="left" rowspan="1" colspan="1"><bold>Phosphorylation sites</bold></th><th valign="top" align="left" rowspan="1" colspan="1"><bold>Process in which were described</bold></th><th valign="top" align="left" rowspan="1" colspan="1"><bold>References</bold></th></tr></thead><tbody><tr><td valign="top" align="left" rowspan="1" colspan="1">2606</td><td valign="top" align="left" rowspan="1" colspan="1">Heat shock protein 60 AT3G23990.1, <italic>Quercus</italic> spp.</td><td valign="top" align="left" rowspan="1" colspan="1">TC33448_39</td><td valign="top" align="left" rowspan="1" colspan="1"><italic>Arabidopsis thaliana</italic></td><td valign="top" align="left" rowspan="1" colspan="1">474 s</td><td valign="top" align="left" rowspan="1" colspan="1">Phosphoproteome characterization of Arabidopsis seedlings shoots and rosette leaves using IMAC and TiO2 phosphopeptide enrichment strategies</td><td valign="top" align="left" rowspan="1" colspan="1">Reiland et al., <xref rid="B37" ref-type="bibr">2009</xref></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">7318</td><td valign="top" align="left" rowspan="1" colspan="1">Phosphoglycerate kinase_AT1G79550.1, <italic>Quercus rubra</italic></td><td valign="top" align="left" rowspan="1" colspan="1">QRU405_58</td><td valign="top" align="left" rowspan="1" colspan="1"><italic>Arabidopsis thaliana</italic></td><td valign="top" align="left" rowspan="1" colspan="1">81S 86T 87S</td><td rowspan="1" colspan="1"></td><td valign="top" align="left" rowspan="1" colspan="1">Reiland et al., <xref rid="B37" ref-type="bibr">2009</xref></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">1711</td><td valign="top" align="left" rowspan="1" colspan="1">Cell division protein ftsH, putative, <italic>Ricinus communis</italic></td><td valign="top" align="left" rowspan="1" colspan="1">B9S304</td><td valign="top" align="left" rowspan="1" colspan="1"><italic>Arabidopsis thaliana</italic></td><td valign="top" align="left" rowspan="1" colspan="1">86T</td><td rowspan="1" colspan="1"></td><td valign="top" align="left" rowspan="1" colspan="1">Reiland et al., <xref rid="B37" ref-type="bibr">2009</xref></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">1303</td><td valign="top" align="left" rowspan="1" colspan="1">DEAD box RNA helicase, <italic>Pisum sativum</italic></td><td valign="top" align="left" rowspan="1" colspan="1">Q8H1A5</td><td valign="top" align="left" rowspan="1" colspan="1"><italic>Arabidopsis thaliana</italic></td><td valign="top" align="left" rowspan="1" colspan="1">76S 86S 85Y 105S 716S 723S</td><td rowspan="1" colspan="1"></td><td valign="top" align="left" rowspan="1" colspan="1">Reiland et al., <xref rid="B37" ref-type="bibr">2009</xref></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">4107</td><td valign="top" align="left" rowspan="1" colspan="1">Unknown protein</td><td valign="top" align="left" rowspan="1" colspan="1">A9PFJ3</td><td valign="top" align="left" rowspan="1" colspan="1"><italic>Oryza sativa</italic></td><td valign="top" align="left" rowspan="1" colspan="1">235S 261S</td><td valign="top" align="left" rowspan="1" colspan="1">Large-scale analysis of rice phosphorylation sites from non-stimulated suspension-cultured rice cells</td><td valign="top" align="left" rowspan="1" colspan="1">Nakagami et al., <xref rid="B35" ref-type="bibr">2010</xref></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">3612</td><td valign="top" align="left" rowspan="1" colspan="1">Glutamate decarboxylase_AT2G02010.1, <italic>Quercus</italic> spp.</td><td valign="top" align="left" rowspan="1" colspan="1">TC19169_41</td><td valign="top" align="left" rowspan="1" colspan="1"><italic>Arabidopsis thaliana</italic></td><td valign="top" align="left" rowspan="1" colspan="1">8S 10S 13S</td><td rowspan="1" colspan="1"></td><td valign="top" align="left" rowspan="1" colspan="1">Nakagami et al., <xref rid="B35" ref-type="bibr">2010</xref></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">4304</td><td valign="top" align="left" rowspan="1" colspan="1">Glucose-1-phosphate adenylyltransferase, <italic>Vitis vinifera</italic></td><td valign="top" align="left" rowspan="1" colspan="1">D7TDB6</td><td valign="top" align="left" rowspan="1" colspan="1"><italic>Arabidopsis thaliana</italic></td><td valign="top" align="left" rowspan="1" colspan="1">77S</td><td rowspan="1" colspan="1"></td><td valign="top" align="left" rowspan="1" colspan="1">Nakagami et al., <xref rid="B35" ref-type="bibr">2010</xref></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">3609</td><td valign="top" align="left" rowspan="1" colspan="1">Phosphoglycerate mutase_AT1G09780.1, <italic>Quercus petraea</italic></td><td valign="top" align="left" rowspan="1" colspan="1">QP1063_77</td><td valign="top" align="left" rowspan="1" colspan="1"><italic>Arabidopsis thaliana</italic></td><td valign="top" align="left" rowspan="1" colspan="1">82 s</td><td valign="top" align="left" rowspan="1" colspan="1">Phosphoproteome role in tobacco pollen activated <italic>in vitro</italic> and large-scale phosphoproteomics</td><td valign="top" align="left" rowspan="1" colspan="1">Nakagami et al., <xref rid="B35" ref-type="bibr">2010</xref>; Fíla et al., <xref rid="B12" ref-type="bibr">2012</xref></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">3507</td><td valign="top" align="left" rowspan="1" colspan="1">Beta glucosidase 17_AT2G44480.1, <italic>Quercus</italic> spp.</td><td valign="top" align="left" rowspan="1" colspan="1">QRO15180_40</td><td valign="top" align="left" rowspan="1" colspan="1"><italic>Medicago truncatula</italic></td><td valign="top" align="left" rowspan="1" colspan="1">82 T</td><td valign="top" align="left" rowspan="1" colspan="1">Large-scale phosphoproteomics analysis in roots</td><td valign="top" align="left" rowspan="1" colspan="1">Grimsrud et al., <xref rid="B17" ref-type="bibr">2010</xref></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">4610</td><td valign="top" align="left" rowspan="1" colspan="1">Pyruvate decarboxylase, <italic>Prunus armeniaca</italic></td><td valign="top" align="left" rowspan="1" colspan="1">B0ZS79</td><td valign="top" align="left" rowspan="1" colspan="1"><italic>Nicotiana tabacum</italic></td><td valign="top" align="left" rowspan="1" colspan="1">380T</td><td valign="top" align="left" rowspan="1" colspan="1">Phosphoproteome role in tobacco pollen activated <italic>in vitro</italic></td><td valign="top" align="left" rowspan="1" colspan="1">Fíla et al., <xref rid="B12" ref-type="bibr">2012</xref></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">5604</td><td valign="top" align="left" rowspan="1" colspan="1">Pyruvate decarboxylase, Putative, <italic>Ricinus communis</italic></td><td valign="top" align="left" rowspan="1" colspan="1">gi55563082</td><td valign="top" align="left" rowspan="1" colspan="1"><italic>Nicotiana tabacum</italic></td><td valign="top" align="left" rowspan="1" colspan="1">380 T</td><td rowspan="1" colspan="1"></td><td valign="top" align="left" rowspan="1" colspan="1">Fíla et al., <xref rid="B12" ref-type="bibr">2012</xref></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">8106</td><td valign="top" align="left" rowspan="1" colspan="1">AT4G39230.1_ NmrA-like negative transcriptional regulator family protein, <italic>Quercus robur</italic></td><td valign="top" align="left" rowspan="1" colspan="1">QRO2324_17</td><td valign="top" align="left" rowspan="1" colspan="1"><italic>Medicago truncatula</italic></td><td valign="top" align="left" rowspan="1" colspan="1">171 T</td><td valign="top" align="left" rowspan="1" colspan="1">Integrated large-scale approach to investigate changes in the phosphoproteome, proteome, and transcriptome that occur 1 h after Nod factors treatment in <italic>Medicago truncatula</italic></td><td valign="top" align="left" rowspan="1" colspan="1">Rose et al., <xref rid="B42" ref-type="bibr">2012b</xref></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">4303</td><td valign="top" align="left" rowspan="1" colspan="1">S-adenosylmethionine synthase 2, <italic>Elaeagnus umbellate</italic></td><td valign="top" align="left" rowspan="1" colspan="1">Q9AT55</td><td valign="top" align="left" rowspan="1" colspan="1"><italic>Medicago truncatula</italic></td><td valign="top" align="left" rowspan="1" colspan="1">131 s 266 s</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">4103</td><td valign="top" align="left" rowspan="1" colspan="1">Glutathione S-transferase omega_D6BR66, <italic>Quercus</italic> spp.</td><td valign="top" align="left" rowspan="1" colspan="1">TC18312_19</td><td valign="top" align="left" rowspan="1" colspan="1"><italic>Medicago truncatula</italic></td><td valign="top" align="left" rowspan="1" colspan="1">10 T</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">4108</td><td valign="top" align="left" rowspan="1" colspan="1">Aluminum induced protein with YGL and LRDR motifs_AT3G22850.1, <italic>Quercus</italic> spp.</td><td valign="top" align="left" rowspan="1" colspan="1">TC18137_21</td><td valign="top" align="left" rowspan="1" colspan="1"><italic>Arabidopsis thaliana</italic></td><td valign="top" align="left" rowspan="1" colspan="1">215S 216S 240S</td><td valign="top" align="left" rowspan="1" colspan="1">Whole cell suspension line, seedlings and seed maturation of rapessed, Arabidopsis and soybean phosphoproteome</td><td valign="top" align="left" rowspan="1" colspan="1">Sugiyama et al., <xref rid="B47" ref-type="bibr">2008</xref>; Meyer et al., <xref rid="B34" ref-type="bibr">2012</xref></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">6003</td><td valign="top" align="left" rowspan="1" colspan="1">Manganese superoxide dismutase 1_AT3G10920.1, <italic>Quercus</italic> spp.</td><td valign="top" align="left" rowspan="1" colspan="1">TC29211_11</td><td valign="top" align="left" rowspan="1" colspan="1"><italic>Glycine max</italic></td><td valign="top" align="left" rowspan="1" colspan="1">173S</td><td valign="top" align="left" rowspan="1" colspan="1">Analysis of seed maturation in Arabidopsis, rapeseed, and Soybean</td><td valign="top" align="left" rowspan="1" colspan="1">Meyer et al., <xref rid="B34" ref-type="bibr">2012</xref></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">7502</td><td valign="top" align="left" rowspan="1" colspan="1">Pyrophosphate-dependent phosphofructokinase beta subunit. <italic>Citrus sinensis</italic> × <italic>Citrus trifoliata</italic></td><td valign="top" align="left" rowspan="1" colspan="1">A9YVC9</td><td valign="top" align="left" rowspan="1" colspan="1"><italic>Arabidopsis thaliana</italic></td><td valign="top" align="left" rowspan="1" colspan="1">12T 16S</td><td valign="top" align="left" rowspan="1" colspan="1">Nuclear phosphoproteinsanalysis of Arabidopsis</td><td valign="top" align="left" rowspan="1" colspan="1">Jones et al., <xref rid="B20" ref-type="bibr">2009</xref>; Reiland et al., <xref rid="B37" ref-type="bibr">2009</xref></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">5003</td><td valign="top" align="left" rowspan="1" colspan="1">Putative uncharacterized protein (Glutathione-s-transferase theta_B9T0U8),<italic>Vitis vinífera</italic></td><td valign="top" align="left" rowspan="1" colspan="1">D7TP00</td><td valign="top" align="left" rowspan="1" colspan="1"><italic>Arabidopsis thaliana</italic></td><td valign="top" align="left" rowspan="1" colspan="1">12 s</td><td valign="top" align="left" rowspan="1" colspan="1">Large-scale phosphoproteome analysis of Arabidopsis cell suspension line,</td><td valign="top" align="left" rowspan="1" colspan="1">Sugiyama et al., <xref rid="B47" ref-type="bibr">2008</xref></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">7702</td><td valign="top" align="left" rowspan="1" colspan="1">5-methyltetrahydropteroyltriglutamate homocysteine methyltransferase-like, <italic>Solanum lycopersicum</italic></td><td valign="top" align="left" rowspan="1" colspan="1">460407874</td><td valign="top" align="left" rowspan="1" colspan="1"><italic>Arabidopsis thaliana</italic></td><td valign="top" align="left" rowspan="1" colspan="1">698 Y 702 S-</td><td rowspan="1" colspan="1"></td><td valign="top" align="left" rowspan="1" colspan="1">Sugiyama et al., <xref rid="B47" ref-type="bibr">2008</xref></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">3103</td><td valign="top" align="left" rowspan="1" colspan="1">Putative cyclase family protein, <italic>Arachis hypogaea</italic></td><td valign="top" align="left" rowspan="1" colspan="1">C0L2U1</td><td valign="top" align="left" rowspan="1" colspan="1">No hits</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr></tbody></table></table-wrap><p>The identified proteins were grouped into functional categories based on the KEGG pathways database (Table <xref ref-type="table" rid="T2">2</xref>): carbohydrate and amino acid metabolism, defense, protein folding and oxidation-reduction processes.</p></sec></sec><sec sec-type="discussion" id="s4"><title>Discussion</title><p>As a preliminary step in the phosphoproteome analysis during the seed germination and early seedling growth processes of a non-orthodox sp. <italic>Q. ilex</italic>, a multiplex (SYPRO-Ruby and Pro-Q DPS) staining of high-resolution 2-DE gels was used. With this protocol it was possible to detect changes in protein-abundance and/or phosphorylation status, identifying, at the same time, candidate phosphoproteins. This simple technique could be a good complementary alternative to the enrichment protocols used in the search for phosphoprotein (Subba et al., <xref rid="B46" ref-type="bibr">2013</xref>; Han et al., <xref rid="B18" ref-type="bibr">2014</xref>; Li et al., <xref rid="B28" ref-type="bibr">2015</xref>). Phosphoprotein enrichment apart from providing, as Pro-Q staining does, false positives, involves excessive manipulation of the sample that results in protein and PTM losses and possible biases. It is true that phosphoprotein validation requires the identification of the phosphorylated peptide, this not being possible or being moret difficult through the MALDI-TOF-TOF MS strategy employed in this work (Thingholm et al., <xref rid="B49" ref-type="bibr">2009</xref>). The protocol presented suffers from the inherent limitations of the 2-DE coupled to the MALDI-TOF-TOF strategy, such as the possible existence and identification of commigrating spots. In at least one case, that of the cyclase, a phosphopeptide was identified, thus confirming its phosphoprotein nature. In any case, rather than identifying the site of phosphorylation, our objective was to search for putative phosphoroteins that showed changes in the phosphorylation status and interpreted those changes from a biological point of view. Different evidence confirmed that most of the Pro-Q–DPS stained spots identified corresponded to real phosphoproteins. Thus: (i) they are Pro-Q stained; (ii) they were only considered if the Pro-Q DPS/SYPRO-Ruby volume ratios were higher than those obtained for a negative control, non-phosphorylated markers (β-galactosidase and serum albumin in this work) and with a ratio equal to or higher than to those obtained for phosphorylated ovoalbumin used as a positive control (Agrawal and Thelen, <xref rid="B2" ref-type="bibr">2006</xref>); (iii) phosphoprotein orthologs have been reported for four different plant species, including <italic>A. thaliana, M. truncatula, N. tabacum</italic>, and <italic>G. max;</italic> (iv) biological interpretation of the data, as discussed below, fits in very well with what is known about the regulation of the identified proteins by phosphorylation. The percentage of phosphorylated proteins detected in our experimental system was of 46%, with similar figures reported for chickpea seedlings (300, Subba et al., <xref rid="B46" ref-type="bibr">2013</xref>) but lower than those reported for germinating rice seeds (500, Han et al., <xref rid="B18" ref-type="bibr">2014</xref>). This could be due to the different methodological approaches and the experimental system used, rather than the system itself or the biological process used or the experimental conditions rather than differences in the number of detectable phosphorylated proteins.</p><p>The percentage of protein identification was lower than that obtained in Coomassie stained spots (Valero Galván et al., <xref rid="B52" ref-type="bibr">2011</xref>, <xref rid="B53" ref-type="bibr">2012b</xref>), this being related to the amount of protein present beyond the absence of sequences for <italic>Quercus</italic> in databases. The most important functional categories are discussed. In this work, we paid special attention to those proteins that showed variations in the phosphorylation pattern with no changes in protein abundance, so that they were supposed to be regulated at the post-translational levels. Independent (protein abundance or phosphorylation pattern) or simultaneous, multiplex (protein abundance and phosphorylation pattern) proteomics analysis by using a similar (bottom-up, 2-DE based) strategy has been used in the analysis of mature orthodox seed and seed germination process in model and crop plant species, including <italic>Arabidopsis</italic>, rice, soybean, rapeseed and maize (Lu et al., <xref rid="B31" ref-type="bibr">2008</xref>; Meyer et al., <xref rid="B34" ref-type="bibr">2012</xref>; Han et al., <xref rid="B18" ref-type="bibr">2014</xref>).</p><p>Three proteins belonged to the <italic>carbohydrate metabolism</italic> category: pyrophosphate-dependent phosphofructokinase (PPi-PFK, spot 7502), phosphoglycerate kinase (PGK, spot 7318) and glucose-1-phosphate adenylyltransferase (AGP, spot 4304) (Table <xref ref-type="table" rid="T2">2</xref> and Figure <xref ref-type="fig" rid="F3">3</xref>). PPi-PFK is a cytosolic enzyme that catalyzes the phosphorylation of fructose-6-phosphate to fructose-1,6-bisphosphate in the glycolytic direction, using inorganic pyrophosphate as the phosphoryl donor. This process makes fructose flow into glycolysis to provide energy. PGK catalyzes the conversion of 1,3-diphosphoglycerate to 3-phosphoglycerate, the first substrate-level phosphorylation reaction in the glycolytic pathway for production of ATP. AGP catalyzes the synthesis of ADP-glucose, which is the active glucoside for starch synthesis (Figure <xref ref-type="fig" rid="F3">3</xref>). Overall, the phosphorylation states of these three enzymes increased throughout the germination process. Phosphorylation modification of many glycolytic enzymes has been reported to cause a significant increase in enzyme activity (Li et al., <xref rid="B29" ref-type="bibr">2011</xref>), incrementing the glycolysis rate and the generation of energy to supply the needs of the developing seedling. These results are in agreement with previous studies on rice germination and seedling (Nakagami et al., <xref rid="B35" ref-type="bibr">2010</xref>; Chen et al., <xref rid="B9" ref-type="bibr">2014</xref>; Han et al., <xref rid="B18" ref-type="bibr">2014</xref>). An increased glycolytic activity in germinating <italic>Q. ilex</italic> seeds is supported by a decrease in sucrose content (Romero-Rodríguez, <xref rid="B39" ref-type="bibr">2015</xref>).</p><fig id="F3" position="float"><label>Figure 3</label><caption><p><bold>Carbohydrate metabolic pathway constructed with proteins whose phosphorylation changed</bold>. Proteins are represented by a green circle. The figures represent the normalized spots volume vs. analyzed stages of each protein, also shown is the spot volume in the stages analyzed.</p></caption><graphic xlink:href="fpls-06-00620-g0003"></graphic></fig><p>On the contrary, for enzymes of the amino acid metabolism (Glutamate decarboxylase, spot 3612) and chaperones (Heat shock protein 60, spot 2606) a decrease in their phosphorylation signal was observed (Table <xref ref-type="table" rid="T2">2</xref>). Glutamate decarboxylase (GDC) catalyzes the decarboxylation of glutamate to GABA, a non-protein amino acid involved in stress tolerances that accumulates in germinating seeds of rice and tomato (Taji et al., <xref rid="B48" ref-type="bibr">2002</xref>; Leitner et al., <xref rid="B26" ref-type="bibr">2011</xref>). Some isoforms of this enzyme are inhibited by phosphorylation (Bao et al., <xref rid="B5" ref-type="bibr">1995</xref>). If applicable to GDC, the reduction in its phosphorylation status observed here might imply an increase in the activity of this enzyme, to eliminate the excess of glutamate and glutamine originated by the high rates of stored proteins degradation occurring during germination.</p><p>The phosphorylation status of heat shock proteins (HSPs), involved in <italic>protein folding</italic>, has been described decreasing during rice germination (Han et al., <xref rid="B18" ref-type="bibr">2014</xref>). In agreement with that, HSP60 showed high levels of phosphorylation in non-imbibed (0 h after imbibition) and germinated seeds (24 h after imbibition).</p><p>In conclusion, over 200 putative phosphoproteins spots were detected in our analysis. Among them, 20 proteins exhibited significant changes in their phosphorylation status, seven of which were identified. Identified enzymes of the glycolytic (pyrophosphate-dependent phosphofructokinase and phosphoglycerate kinase) and amino acid metabolic pathways (glutamate decarboxylase) and protein folding (heat shock protein 60) did not change in abundance during germination and growth but their phosphorylation status increased suggesting regulation at the post-translational level. Alterations in the phosphorylation status of proteins related to glycolysis and amino acid metabolism are in agreement considering that these pathways must increase from mature seeds to germinated seeds and seedling. To test these hypotheses it is necessary identify the phosphorylation sites, but this work constitutes an initiation in the study of the molecular mechanism involved in <italic>Q. ilex</italic> seed germination. The phosphoproteome analysis suggested that the metabolic machinery present in the recalcitrant seeds receives a signal to activate and resume/summarize the most important metabolic pathways in <italic>Q. ilex</italic> to start the germination and the establishment of the seedlings. In orthodox seeds, changes in abundance, together with differences in their phosphorylation status, were observed for these enzymes. Thus, this is one of the differences between orthodox and non-orthodox seeds that may explain their different behavior (Han et al., <xref rid="B18" ref-type="bibr">2014</xref>).</p><sec><title>Conflict of interest statement</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></sec></body><back><sec sec-type="supplementary-material" id="s5"><title>Supplementary material</title><p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type="uri" xlink:href="http://journal.frontiersin.org/article/10.3389/fpls.2015.00620">http://journal.frontiersin.org/article/10.3389/fpls.2015.00620</ext-link></p><supplementary-material content-type="local-data" id="SM1"><media xlink:href="Table1.XLSX"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="SM2"><media xlink:href="Presentation1.PDF"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec><ref-list><title>References</title><ref id="B1"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Agrawal</surname><given-names>G. K.</given-names></name><name><surname>Thelen</surname><given-names>J. J.</given-names></name></person-group> (<year>2005</year>). <article-title>Development of a simplified, economical polyacrylamide gel staining protocol for phosphoproteins</article-title>. <source>Proteomics</source><volume>5</volume>, <fpage>4684</fpage>–<lpage>4688</lpage>. <pub-id pub-id-type="doi">10.1002/pmic.200500021</pub-id><pub-id pub-id-type="pmid">16267815</pub-id></mixed-citation></ref><ref id="B2"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Agrawal</surname><given-names>G. K.</given-names></name><name><surname>Thelen</surname><given-names>J. J.</given-names></name></person-group> (<year>2006</year>). <article-title>Large scale identification and quantitative profiling of phosphoproteins expressed during seed filling in oilseed rape</article-title>. <source>Mol. Cell. Proteomics</source><volume>5</volume>, <fpage>2044</fpage>–<lpage>2059</lpage>. <pub-id pub-id-type="doi">10.1074/mcp.M600084-MCP200</pub-id><pub-id pub-id-type="pmid">16825184</pub-id></mixed-citation></ref><ref id="B3"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Alonso</surname><given-names>J.</given-names></name><name><surname>Zapata</surname><given-names>C.</given-names></name></person-group> (<year>2014</year>). <article-title>Evidence for phosphorylation of the major seed storage protein of the common bean and its phosphorylation-dependent degradation during germination</article-title>. <source>Plant Mol. Biol.</source><volume>84</volume>, <fpage>415</fpage>–<lpage>428</lpage>. <pub-id pub-id-type="doi">10.1007/s11103-013-0141-1</pub-id><pub-id pub-id-type="pmid">24142381</pub-id></mixed-citation></ref><ref id="B4"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Balbuena</surname><given-names>T. S.</given-names></name><name><surname>Jo</surname><given-names>L.</given-names></name><name><surname>Pieruzzi</surname><given-names>F. P.</given-names></name><name><surname>Dias</surname><given-names>L. L. C.</given-names></name><name><surname>Silveira</surname><given-names>V.</given-names></name><name><surname>Santa-Catarina</surname><given-names>C.</given-names></name><etal></etal></person-group>. (<year>2011</year>). <article-title>Differential proteome analysis of mature and germinated embryos of <italic>Araucaria angustifolia</italic></article-title>. <source>Phytochemistry</source><volume>72</volume>, <fpage>302</fpage>–<lpage>311</lpage>. <pub-id pub-id-type="doi">10.1016/j.phytochem.2010.12.007</pub-id><pub-id pub-id-type="pmid">21276992</pub-id></mixed-citation></ref><ref id="B5"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Bao</surname><given-names>J.</given-names></name><name><surname>Cheung</surname><given-names>W. Y.</given-names></name><name><surname>Jy</surname><given-names>W</given-names></name></person-group>. (<year>1995</year>). <article-title>Brain L-glutamate decarboxylase. Inhibition by phosphorylation and activation by dephosphorylation.</article-title><source>J. Biol. Chem.</source><volume>270</volume>, <fpage>6464</fpage>–<lpage>6467</lpage>. <pub-id pub-id-type="pmid">7896780</pub-id></mixed-citation></ref><ref id="B6"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Berggren</surname><given-names>K.</given-names></name><name><surname>Chernokalskaya</surname><given-names>E.</given-names></name><name><surname>Steinberg</surname><given-names>T. H.</given-names></name><name><surname>Kemper</surname><given-names>C.</given-names></name><name><surname>Lopez</surname><given-names>M. F.</given-names></name><name><surname>Diwu</surname><given-names>Z.</given-names></name><etal></etal></person-group>. (<year>2000</year>). <article-title>Background-free, high sensitivity staining of proteins in one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gels using a luminescent ruthenium complex</article-title>. <source>Electrophoresis</source><volume>21</volume>, <fpage>2509</fpage>–<lpage>2521</lpage>. <pub-id pub-id-type="doi">10.1002/1522-2683(20000701)21:12<2509::AID-ELPS2509>3.0.CO;2-9</pub-id><pub-id pub-id-type="pmid">10939466</pub-id></mixed-citation></ref><ref id="B7"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Bond</surname><given-names>A. E.</given-names></name><name><surname>Row</surname><given-names>P. E.</given-names></name><name><surname>Dudley</surname><given-names>E.</given-names></name></person-group> (<year>2011</year>). <article-title>Post-translation modification of proteins; methodologies and applications in plant sciences</article-title>. <source>Phytochemistry</source><volume>72</volume>, <fpage>975</fpage>–<lpage>996</lpage>. <pub-id pub-id-type="doi">10.1016/j.phytochem.2011.01.029</pub-id><pub-id pub-id-type="pmid">21353264</pub-id></mixed-citation></ref><ref id="B8"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Bradford</surname><given-names>M. M.</given-names></name></person-group> (<year>1976</year>). <article-title>A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding</article-title>. <source>Anal. Biochem.</source><volume>72</volume>, <fpage>248</fpage>–<lpage>254</lpage>. <pub-id pub-id-type="doi">10.1016/0003-2697(76)90527-3</pub-id><pub-id pub-id-type="pmid">942051</pub-id></mixed-citation></ref><ref id="B9"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Chen</surname><given-names>F.</given-names></name><name><surname>Jiang</surname><given-names>L.</given-names></name><name><surname>Zheng</surname><given-names>J.</given-names></name><name><surname>Huang</surname><given-names>R.</given-names></name><name><surname>Wang</surname><given-names>H.</given-names></name><name><surname>Hong</surname><given-names>Z.</given-names></name><etal></etal></person-group>. (<year>2014</year>). <article-title>Identification of differentially expressed proteins and phosphorylated proteins in rice seedlings in response to strigolactone treatment</article-title>. <source>PLoS ONE</source><volume>9</volume>:<fpage>e93947</fpage>. <pub-id pub-id-type="doi">10.1371/journal.pone.0093947</pub-id><pub-id pub-id-type="pmid">24699514</pub-id></mixed-citation></ref><ref id="B10"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Conesa</surname><given-names>A.</given-names></name><name><surname>Götz</surname><given-names>S.</given-names></name><name><surname>García-Gómez</surname><given-names>J. M.</given-names></name><name><surname>Terol</surname><given-names>J.</given-names></name><name><surname>Talón</surname><given-names>M.</given-names></name><name><surname>Robles</surname><given-names>M.</given-names></name></person-group> (<year>2005</year>). <article-title>Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research</article-title>. <source>Bioinformatics</source><volume>21</volume>, <fpage>3674</fpage>–<lpage>3676</lpage>. <pub-id pub-id-type="doi">10.1093/bioinformatics/bti610</pub-id><pub-id pub-id-type="pmid">16081474</pub-id></mixed-citation></ref><ref id="B11"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Cutler</surname><given-names>S. R.</given-names></name><name><surname>Rodriguez</surname><given-names>P. L.</given-names></name><name><surname>Finkelstein</surname><given-names>R. R.</given-names></name><name><surname>Abrams</surname><given-names>S. R.</given-names></name></person-group> (<year>2010</year>). <article-title>Abscisic acid: emergence of a core signaling network</article-title>. <source>Annu. Rev. Plant Biol.</source><volume>61</volume>, <fpage>651</fpage>–<lpage>679</lpage>. <pub-id pub-id-type="doi">10.1146/annurev-arplant-042809-112122</pub-id><pub-id pub-id-type="pmid">20192755</pub-id></mixed-citation></ref><ref id="B12"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Fíla</surname><given-names>J.</given-names></name><name><surname>Matros</surname><given-names>A.</given-names></name><name><surname>Radau</surname><given-names>S.</given-names></name><name><surname>Zahedi</surname><given-names>R. P.</given-names></name><name><surname>C̀apková</surname><given-names>V.</given-names></name><name><surname>Mock</surname><given-names>H.-P.</given-names></name><etal></etal></person-group>. (<year>2012</year>). <article-title>Revealing phosphoproteins playing role in tobacco pollen activated <italic>in vitro</italic></article-title>. <source>Proteomics</source><volume>12</volume>, <fpage>3229</fpage>–<lpage>3250</lpage>. <pub-id pub-id-type="doi">10.1002/pmic.201100318</pub-id><pub-id pub-id-type="pmid">22976843</pub-id></mixed-citation></ref><ref id="B13"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Fujii</surname><given-names>H.</given-names></name><name><surname>Chinnusamy</surname><given-names>V.</given-names></name><name><surname>Rodrigues</surname><given-names>A.</given-names></name><name><surname>Rubio</surname><given-names>S.</given-names></name><name><surname>Antoni</surname><given-names>R.</given-names></name><name><surname>Park</surname><given-names>S.-Y.</given-names></name><etal></etal></person-group>. (<year>2009</year>). <article-title><italic>In vitro</italic> reconstitution of an abscisic acid signalling pathway</article-title>. <source>Nature</source><volume>462</volume>, <fpage>660</fpage>–<lpage>664</lpage>. <pub-id pub-id-type="doi">10.1038/nature08599</pub-id><pub-id pub-id-type="pmid">19924127</pub-id></mixed-citation></ref><ref id="B14"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Gallego</surname><given-names>F. J.</given-names></name><name><surname>De Algaba</surname><given-names>A. P.</given-names></name><name><surname>Fernandez-Escobar</surname><given-names>R.</given-names></name></person-group> (<year>1999</year>). <article-title>Etiology of oak decline in Spain</article-title>. <source>Eur. J. For. Pathol.</source><volume>29</volume>, <fpage>17</fpage>–<lpage>27</lpage>. <pub-id pub-id-type="doi">10.1046/j.1439-0329.1999.00128.x</pub-id></mixed-citation></ref><ref id="B15"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Gao</surname><given-names>J.</given-names></name><name><surname>Agrawal</surname><given-names>G. K.</given-names></name><name><surname>Thelen</surname><given-names>J. J.</given-names></name><name><surname>Xu</surname><given-names>D.</given-names></name></person-group> (<year>2009</year>). <article-title>P3DB: a plant protein phosphorylation database</article-title>. <source>Nucleic Acids Res.</source><volume>37</volume>, <fpage>D960</fpage>–<lpage>D962</lpage>. <pub-id pub-id-type="doi">10.1093/nar/gkn733</pub-id><pub-id pub-id-type="pmid">18931372</pub-id></mixed-citation></ref><ref id="B16"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Görg</surname><given-names>A.</given-names></name><name><surname>Weiss</surname><given-names>W.</given-names></name><name><surname>Dunn</surname><given-names>M. J.</given-names></name></person-group> (<year>2004</year>). <article-title>Current two-dimensional electrophoresis technology for proteomics</article-title>. <source>Proteomics</source><volume>4</volume>, <fpage>3665</fpage>–<lpage>3685</lpage>. <pub-id pub-id-type="doi">10.1002/pmic.200401031</pub-id><pub-id pub-id-type="pmid">15543535</pub-id></mixed-citation></ref><ref id="B17"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Grimsrud</surname><given-names>P. A.</given-names></name><name><surname>Den Os</surname><given-names>D.</given-names></name><name><surname>Wenger</surname><given-names>C. D.</given-names></name><name><surname>Swaney</surname><given-names>D. L.</given-names></name><name><surname>Schwartz</surname><given-names>D.</given-names></name><name><surname>Sussman</surname><given-names>M. R.</given-names></name><etal></etal></person-group>. (<year>2010</year>). <article-title>Large-scale phosphoprotein analysis in <italic>Medicago truncatula</italic> roots provides insight into <italic>in vivo</italic> kinase activity in legumes.</article-title><source>Plant Physiol.</source><volume>152</volume>, <fpage>19</fpage>–<lpage>28</lpage>. <pub-id pub-id-type="doi">10.1104/pp.109.149625</pub-id><pub-id pub-id-type="pmid">19923235</pub-id></mixed-citation></ref><ref id="B18"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Han</surname><given-names>C.</given-names></name><name><surname>Wang</surname><given-names>K.</given-names></name><name><surname>Yang</surname><given-names>P.</given-names></name></person-group> (<year>2014</year>). <article-title>Gel-based comparative phosphoproteomic analysis on rice embryo during germination</article-title>. <source>Plant Cell Physiol</source>. <volume>55</volume>, <fpage>1376</fpage>–<lpage>1394</lpage>. <pub-id pub-id-type="doi">10.1093/pcp/pcu060</pub-id><pub-id pub-id-type="pmid">24793751</pub-id></mixed-citation></ref><ref id="B19"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Hunt</surname><given-names>L.</given-names></name><name><surname>Holdsworth</surname><given-names>M. J.</given-names></name><name><surname>Gray</surname><given-names>J. E.</given-names></name></person-group> (<year>2007</year>). <article-title>Nicotinamidase activity is important for germination</article-title>. <source>Plant J.</source><volume>51</volume>, <fpage>341</fpage>–<lpage>351</lpage>. <pub-id pub-id-type="doi">10.1111/j.1365-313X.2007.03151.x</pub-id><pub-id pub-id-type="pmid">17587307</pub-id></mixed-citation></ref><ref id="B20"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Jones</surname><given-names>A. M. E.</given-names></name><name><surname>Maclean</surname><given-names>D.</given-names></name><name><surname>Studholme</surname><given-names>D. J.</given-names></name><name><surname>Serna-Sanz</surname><given-names>A.</given-names></name><name><surname>Andreasson</surname><given-names>E.</given-names></name><name><surname>Rathjen</surname><given-names>J. P.</given-names></name><etal></etal></person-group>. (<year>2009</year>). <article-title>Phosphoproteomic analysis of nuclei-enriched fractions from <italic>Arabidopsis thaliana</italic></article-title>. <source>J. Proteomics</source><volume>72</volume>, <fpage>439</fpage>–<lpage>451</lpage>. <pub-id pub-id-type="doi">10.1016/j.jprot.2009.02.004</pub-id><pub-id pub-id-type="pmid">19245862</pub-id></mixed-citation></ref><ref id="B21"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Jorge</surname><given-names>I.</given-names></name><name><surname>Navarro</surname><given-names>R. M.</given-names></name><name><surname>Lenz</surname><given-names>C.</given-names></name><name><surname>Ariza</surname><given-names>D.</given-names></name><name><surname>Jorrín</surname><given-names>J.</given-names></name></person-group> (<year>2006</year>). <article-title>Variation in the holm oak leaf proteome at different plant developmental stages, between provenances and in response to drought stress</article-title>. <source>Proteomics</source><volume>6</volume>, <fpage>S207</fpage>–<lpage>S214</lpage>. <pub-id pub-id-type="doi">10.1002/pmic.200500364</pub-id><pub-id pub-id-type="pmid">16534744</pub-id></mixed-citation></ref><ref id="B22"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kaufmann</surname><given-names>H.</given-names></name><name><surname>Bailey</surname><given-names>J. E.</given-names></name><name><surname>Fussenegger</surname><given-names>M.</given-names></name></person-group> (<year>2001</year>). <article-title>Use of antibodies for detection of phosphorylated proteins separated by two-dimensional gel electrophoresis</article-title>. <source>Proteomics</source><volume>1</volume>, <fpage>194</fpage>–<lpage>199</lpage>. <pub-id pub-id-type="doi">10.1002/1615-9861(200102)1:2<194::AID-PROT194>3.0.CO;2-K</pub-id><pub-id pub-id-type="pmid">11680866</pub-id></mixed-citation></ref><ref id="B23"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kersten</surname><given-names>B.</given-names></name><name><surname>Agrawal</surname><given-names>G. K.</given-names></name><name><surname>Durek</surname><given-names>P.</given-names></name><name><surname>Neigenfind</surname><given-names>J.</given-names></name><name><surname>Schulze</surname><given-names>W.</given-names></name><name><surname>Walther</surname><given-names>D.</given-names></name><etal></etal></person-group>. (<year>2009</year>). <article-title>Plant phosphoproteomics: an update</article-title>. <source>Proteomics</source><volume>9</volume>, <fpage>964</fpage>–<lpage>988</lpage>. <pub-id pub-id-type="doi">10.1002/pmic.200800548</pub-id><pub-id pub-id-type="pmid">19212952</pub-id></mixed-citation></ref><ref id="B24"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kersten</surname><given-names>B.</given-names></name><name><surname>Agrawal</surname><given-names>G. K.</given-names></name><name><surname>Iwahashi</surname><given-names>H.</given-names></name><name><surname>Rakwal</surname><given-names>R.</given-names></name></person-group> (<year>2006</year>). <article-title>Plant phosphoproteomics: a long road ahead</article-title>. <source>Proteomics</source><volume>6</volume>, <fpage>5517</fpage>–<lpage>5528</lpage>. <pub-id pub-id-type="doi">10.1002/pmic.200600232</pub-id><pub-id pub-id-type="pmid">16991200</pub-id></mixed-citation></ref><ref id="B25"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Laemmli</surname><given-names>U. K.</given-names></name></person-group> (<year>1970</year>). <article-title>Cleavage of structural proteins during the assembly of the head of bacteriophage T4</article-title>. <source>Nature</source><volume>227</volume>, <fpage>680</fpage>–<lpage>685</lpage>. <pub-id pub-id-type="doi">10.1038/227680a0</pub-id><pub-id pub-id-type="pmid">5432063</pub-id></mixed-citation></ref><ref id="B26"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Leitner</surname><given-names>A.</given-names></name><name><surname>Sturm</surname><given-names>M.</given-names></name><name><surname>Lindner</surname><given-names>W.</given-names></name></person-group> (<year>2011</year>). <article-title>Tools for analyzing the phosphoproteome and other phosphorylated biomolecules: a review</article-title>. <source>Anal. Chim. Acta</source><volume>703</volume>, <fpage>19</fpage>–<lpage>30</lpage>. <pub-id pub-id-type="doi">10.1016/j.aca.2011.07.012</pub-id><pub-id pub-id-type="pmid">21843671</pub-id></mixed-citation></ref><ref id="B27"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Li</surname><given-names>H.</given-names></name><name><surname>Wong</surname><given-names>W. S.</given-names></name><name><surname>Zhu</surname><given-names>L.</given-names></name><name><surname>Guo</surname><given-names>H. W.</given-names></name><name><surname>Ecker</surname><given-names>J.</given-names></name><name><surname>Li</surname><given-names>N.</given-names></name></person-group> (<year>2009</year>). <article-title>Phosphoproteomic analysis of ethylene-regulated protein phosphorylation in etiolated seedlings of Arabidopsis mutant ein2 using two-dimensional separations coupled with a hybrid quadrupole time-of-flight mass spectrometer</article-title>. <source>Proteomics</source><volume>9</volume>, <fpage>1646</fpage>–<lpage>1661</lpage>. <pub-id pub-id-type="doi">10.1002/pmic.200800420</pub-id><pub-id pub-id-type="pmid">19253305</pub-id></mixed-citation></ref><ref id="B28"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Li</surname><given-names>J.</given-names></name><name><surname>Silva-Sanchez</surname><given-names>C.</given-names></name><name><surname>Zhang</surname><given-names>T.</given-names></name><name><surname>Chen</surname><given-names>S.</given-names></name><name><surname>Li</surname><given-names>H.</given-names></name></person-group> (<year>2015</year>). <article-title>Phosphoproteomics technologies and applications in plant biology research</article-title>. <source>Front. Plant Sci.</source><volume>6</volume>:<issue>430</issue>. <pub-id pub-id-type="doi">10.3389/fpls.2015.00430</pub-id><pub-id pub-id-type="pmid">26136758</pub-id></mixed-citation></ref><ref id="B29"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Li</surname><given-names>X.</given-names></name><name><surname>Zhuo</surname><given-names>J.</given-names></name><name><surname>Jing</surname><given-names>Y.</given-names></name><name><surname>Liu</surname><given-names>X.</given-names></name><name><surname>Wang</surname><given-names>X.</given-names></name></person-group> (<year>2011</year>). <article-title>Expression of a GALACTINOL SYNTHASE gene is positively associated with desiccation tolerance of <italic>Brassica napus</italic> seeds during development</article-title>. <source>J. Plant Physiol.</source><volume>168</volume>, <fpage>1761</fpage>–<lpage>1770</lpage>. <pub-id pub-id-type="doi">10.1016/j.jplph.2011.04.006</pub-id><pub-id pub-id-type="pmid">21680054</pub-id></mixed-citation></ref><ref id="B30"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Liu</surname><given-names>Y.</given-names></name><name><surname>Liu</surname><given-names>G.</given-names></name><name><surname>Li</surname><given-names>Q.</given-names></name><name><surname>Liu</surname><given-names>Y.</given-names></name><name><surname>Hou</surname><given-names>L.</given-names></name><name><surname>Li</surname><given-names>G.</given-names></name></person-group> (<year>2012</year>). <article-title>Influence of pericarp, cotyledon and inhibitory substances on sharp tooth oak (<italic>Quercus aliena</italic> var. acuteserrata) Germination.</article-title><source>PLoS ONE</source><volume>7</volume>:<fpage>e47682</fpage>. <pub-id pub-id-type="doi">10.1371/journal.pone.0047682</pub-id><pub-id pub-id-type="pmid">23133517</pub-id></mixed-citation></ref><ref id="B31"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lu</surname><given-names>T.-C.</given-names></name><name><surname>Meng</surname><given-names>L.-B.</given-names></name><name><surname>Yang</surname><given-names>C.-P.</given-names></name><name><surname>Liu</surname><given-names>G.-F.</given-names></name><name><surname>Liu</surname><given-names>G.-J.</given-names></name><name><surname>Ma</surname><given-names>W.</given-names></name><etal></etal></person-group>. (<year>2008</year>). <article-title>A shotgun phosphoproteomics analysis of embryos in germinated maize seeds</article-title>. <source>Planta</source><volume>228</volume>, <fpage>1029</fpage>–<lpage>1041</lpage>. <pub-id pub-id-type="doi">10.1007/s00425-008-0805-2</pub-id><pub-id pub-id-type="pmid">18726113</pub-id></mixed-citation></ref><ref id="B32"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Maldonado</surname><given-names>A. M.</given-names></name><name><surname>Echevarría-Zomeño</surname><given-names>S.</given-names></name><name><surname>Jean-Baptiste</surname><given-names>S.</given-names></name><name><surname>Hernández</surname><given-names>M.</given-names></name><name><surname>Jorrín-Novo</surname><given-names>J. V.</given-names></name></person-group> (<year>2008</year>). <article-title>Evaluation of three different protocols of protein extraction for <italic>Arabidopsis thaliana</italic> leaf proteome analysis by two-dimensional electrophoresis</article-title>. <source>J. Proteomics</source><volume>71</volume>, <fpage>461</fpage>–<lpage>472</lpage>. <pub-id pub-id-type="doi">10.1016/j.jprot.2008.06.012</pub-id><pub-id pub-id-type="pmid">18656559</pub-id></mixed-citation></ref><ref id="B33"><mixed-citation publication-type="book"><person-group person-group-type="author"><collab>MAPA</collab></person-group> (<year>2006</year>). <source>Forestación de Tierras Agrícolas</source>. <publisher-loc>Madrid</publisher-loc>: <publisher-name>Ministerio de Agricultura y Pesca</publisher-name>.</mixed-citation></ref><ref id="B34"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Meyer</surname><given-names>L. J.</given-names></name><name><surname>Gao</surname><given-names>J.</given-names></name><name><surname>Xu</surname><given-names>D.</given-names></name><name><surname>Thelen</surname><given-names>J. J.</given-names></name></person-group> (<year>2012</year>). <article-title>Phosphoproteomic analysis of seed maturation in Arabidopsis, rapeseed, and soybean</article-title>. <source>Plant Physiol.</source><volume>159</volume>, <fpage>517</fpage>–<lpage>528</lpage>. <pub-id pub-id-type="doi">10.1104/pp.111.191700</pub-id><pub-id pub-id-type="pmid">22440515</pub-id></mixed-citation></ref><ref id="B35"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Nakagami</surname><given-names>H.</given-names></name><name><surname>Sugiyama</surname><given-names>N.</given-names></name><name><surname>Mochida</surname><given-names>K.</given-names></name><name><surname>Daudi</surname><given-names>A.</given-names></name><name><surname>Yoshida</surname><given-names>Y.</given-names></name><name><surname>Toyoda</surname><given-names>T.</given-names></name><etal></etal></person-group>. (<year>2010</year>). <article-title>Large-scale comparative phosphoproteomics identifies conserved phosphorylation sites in plants</article-title>. <source>Plant Physiol.</source><volume>153</volume>, <fpage>1161</fpage>–<lpage>1174</lpage>. <pub-id pub-id-type="doi">10.1104/pp.110.157347</pub-id><pub-id pub-id-type="pmid">20466843</pub-id></mixed-citation></ref><ref id="B36"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Pulido</surname><given-names>F. J.</given-names></name><name><surname>DíAz</surname><given-names>M.</given-names></name><name><surname>Hidalgo De Trucios</surname><given-names>S. J.</given-names></name></person-group> (<year>2001</year>). <article-title>Size structure and regeneration of Spanish holm oak <italic>Quercus ilex</italic> forests and dehesas: effects of agroforestry use on their long-term sustainability</article-title>. <source>For. Ecol. Manage.</source><volume>146</volume>, <fpage>1</fpage>–<lpage>13</lpage>. <pub-id pub-id-type="doi">10.1016/S0378-1127(00)00443-6</pub-id></mixed-citation></ref><ref id="B37"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Reiland</surname><given-names>S.</given-names></name><name><surname>Messerli</surname><given-names>G.</given-names></name><name><surname>Baerenfaller</surname><given-names>K.</given-names></name><name><surname>Gerrits</surname><given-names>B.</given-names></name><name><surname>Endler</surname><given-names>A.</given-names></name><name><surname>Grossmann</surname><given-names>J.</given-names></name><etal></etal></person-group>. (<year>2009</year>). <article-title>Large-scale Arabidopsis phosphoproteome profiling reveals novel chloroplast kinase substrates and phosphorylation networks</article-title>. <source>Plant Physiol.</source><volume>150</volume>, <fpage>889</fpage>–<lpage>903</lpage>. <pub-id pub-id-type="doi">10.1104/pp.109.138677</pub-id><pub-id pub-id-type="pmid">19376835</pub-id></mixed-citation></ref><ref id="B38"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Rigbolt</surname><given-names>K. T. G.</given-names></name><name><surname>Blagoev</surname><given-names>B.</given-names></name></person-group> (<year>2012</year>). <article-title>Quantitative phosphoproteomics to characterize signaling networks</article-title>. <source>Semin. Cell Dev. Biol.</source><volume>23</volume>, <fpage>863</fpage>–<lpage>871</lpage>. <pub-id pub-id-type="doi">10.1016/j.semcdb.2012.05.006</pub-id><pub-id pub-id-type="pmid">22677334</pub-id></mixed-citation></ref><ref id="B39"><mixed-citation publication-type="book"><person-group person-group-type="author"><name><surname>Romero-Rodríguez</surname><given-names>M. C.</given-names></name></person-group> (<year>2015</year>). <source>Integrated “Omics” Approaches to Study Non-orthodox Seed Germination: The Case of Holm Oak (Quercus ilex Subsp. Ballota [Desf.] Samp.)</source>. <publisher-name>Doctor of Philosophy, University of Cordoba</publisher-name>.</mixed-citation></ref><ref id="B40"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Romero-Rodriguez</surname><given-names>M. C.</given-names></name><name><surname>Pascual</surname><given-names>J.</given-names></name><name><surname>Valledor</surname><given-names>L.</given-names></name><name><surname>Jorrin-Novo</surname><given-names>J.</given-names></name></person-group> (<year>2014</year>). <article-title>Improving the quality of protein identification in non-model species. Characterization of <italic>Quercus ilex</italic> seed and <italic>Pinus radiata</italic> needle proteomes by using SEQUEST and custom databases.</article-title><source>J. Proteomics</source><volume>105</volume>, <fpage>85</fpage>–<lpage>91</lpage>. <pub-id pub-id-type="doi">10.1016/j.jprot.2014.01.027</pub-id><pub-id pub-id-type="pmid">24508333</pub-id></mixed-citation></ref><ref id="B41"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Rose</surname><given-names>C. M.</given-names></name><name><surname>Venkateshwaran</surname><given-names>M.</given-names></name><name><surname>Grimsrud</surname><given-names>P. A.</given-names></name><name><surname>Westphall</surname><given-names>M. S.</given-names></name><name><surname>Sussman</surname><given-names>M. R.</given-names></name><name><surname>Coon</surname><given-names>J. J.</given-names></name><etal></etal></person-group>. (<year>2012a</year>). <article-title>Medicago phosphoprotein database: a repository for <italic>Medicago truncatula</italic> phosphoprotein data</article-title>. <source>Front. Plant Sci.</source><volume>3</volume>:<issue>122</issue>. <pub-id pub-id-type="doi">10.3389/fpls.2012.00122</pub-id><pub-id pub-id-type="pmid">22701463</pub-id></mixed-citation></ref><ref id="B42"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Rose</surname><given-names>C. M.</given-names></name><name><surname>Venkateshwaran</surname><given-names>M.</given-names></name><name><surname>Volkening</surname><given-names>J. D.</given-names></name><name><surname>Grimsrud</surname><given-names>P. A.</given-names></name><name><surname>Maeda</surname><given-names>J.</given-names></name><name><surname>Bailey</surname><given-names>D. J.</given-names></name><etal></etal></person-group>. (<year>2012b</year>). <article-title>Rapid phosphoproteomic and transcriptomic changes in the rhizobia-legume symbiosis</article-title>. <source>Mol. Cell. Proteomics</source><volume>11</volume>, <fpage>724</fpage>–<lpage>744</lpage>. <pub-id pub-id-type="doi">10.1074/mcp.M112.019208</pub-id><pub-id pub-id-type="pmid">22683509</pub-id></mixed-citation></ref><ref id="B43"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Sghaier-Hammami</surname><given-names>B.</given-names></name><name><surname>Valero-Galvàn</surname><given-names>J.</given-names></name><name><surname>Romero-Rodríguez</surname><given-names>M. C.</given-names></name><name><surname>Navarro-Cerrillo</surname><given-names>R. M.</given-names></name><name><surname>Abdelly</surname><given-names>C.</given-names></name><name><surname>Jorrín-Novo</surname><given-names>J.</given-names></name></person-group> (<year>2013</year>). <article-title>Physiological and proteomics analyses of Holm oak (<italic>Quercus ilex</italic> subsp. ballota [Desf.] Samp.) responses to <italic>Phytophthora cinnamomi</italic>.</article-title><source>Plant Physiol. Biochem.</source><volume>71</volume>, <fpage>191</fpage>–<lpage>202</lpage>. <pub-id pub-id-type="doi">10.1016/j.plaphy.2013.06.030</pub-id><pub-id pub-id-type="pmid">23962806</pub-id></mixed-citation></ref><ref id="B44"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Sharov</surname><given-names>A. A.</given-names></name><name><surname>Dudekula</surname><given-names>D. B.</given-names></name><name><surname>Ko</surname><given-names>M. S. H.</given-names></name></person-group> (<year>2005</year>). <article-title>A web-based tool for principal component and significance analysis of microarray data</article-title>. <source>Bioinformatics</source><volume>21</volume>, <fpage>2548</fpage>–<lpage>2549</lpage>. <pub-id pub-id-type="doi">10.1093/bioinformatics/bti343</pub-id><pub-id pub-id-type="pmid">15734774</pub-id></mixed-citation></ref><ref id="B45"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Shevchenko</surname><given-names>A.</given-names></name><name><surname>Wilm</surname><given-names>M.</given-names></name><name><surname>Vorm</surname><given-names>O.</given-names></name><name><surname>Mann</surname><given-names>M.</given-names></name></person-group> (<year>1996</year>). <article-title>Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels</article-title>. <source>Anal. Chem.</source><volume>68</volume>, <fpage>850</fpage>–<lpage>858</lpage>. <pub-id pub-id-type="doi">10.1021/ac950914h</pub-id><pub-id pub-id-type="pmid">8779443</pub-id></mixed-citation></ref><ref id="B46"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Subba</surname><given-names>P.</given-names></name><name><surname>Barua</surname><given-names>P.</given-names></name><name><surname>Kumar</surname><given-names>R.</given-names></name><name><surname>Datta</surname><given-names>A.</given-names></name><name><surname>Soni</surname><given-names>K. K.</given-names></name><name><surname>Chakraborty</surname><given-names>S.</given-names></name><etal></etal></person-group>. (<year>2013</year>). <article-title>Phosphoproteomic dynamics of chickpea (<italic>Cicer arietinum</italic> L.) reveals shared and distinct components of dehydration response</article-title>. <source>J. Proteome Res.</source><volume>12</volume>, <fpage>5025</fpage>–<lpage>5047</lpage>. <pub-id pub-id-type="doi">10.1021/pr400628j</pub-id><pub-id pub-id-type="pmid">24083463</pub-id></mixed-citation></ref><ref id="B47"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Sugiyama</surname><given-names>N.</given-names></name><name><surname>Nakagami</surname><given-names>H.</given-names></name><name><surname>Mochida</surname><given-names>K.</given-names></name><name><surname>Daudi</surname><given-names>A.</given-names></name><name><surname>Tomita</surname><given-names>M.</given-names></name><name><surname>Shirasu</surname><given-names>K.</given-names></name><etal></etal></person-group>. (<year>2008</year>). <article-title>Large-scale phosphorylation mapping reveals the extent of tyrosine phosphorylation in Arabidopsis</article-title>. <source>Mol. Syst. Biol.</source><volume>4</volume>:<fpage>193</fpage>. <pub-id pub-id-type="doi">10.1038/msb.2008.32</pub-id><pub-id pub-id-type="pmid">18463617</pub-id></mixed-citation></ref><ref id="B48"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Taji</surname><given-names>T.</given-names></name><name><surname>Ohsumi</surname><given-names>C.</given-names></name><name><surname>Iuchi</surname><given-names>S.</given-names></name><name><surname>Seki</surname><given-names>M.</given-names></name><name><surname>Kasuga</surname><given-names>M.</given-names></name><name><surname>Kobayashi</surname><given-names>M.</given-names></name><etal></etal></person-group>. (<year>2002</year>). <article-title>Important roles of drought- and cold-inducible genes for galactinol synthase in stress tolerance in <italic>Arabidopsis thaliana</italic></article-title>. <source>Plant J.</source><volume>29</volume>, <fpage>417</fpage>–<lpage>426</lpage>. <pub-id pub-id-type="doi">10.1046/j.0960-7412.2001.01227.x</pub-id><pub-id pub-id-type="pmid">11846875</pub-id></mixed-citation></ref><ref id="B49"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Thingholm</surname><given-names>T. E.</given-names></name><name><surname>Jensen</surname><given-names>O. N.</given-names></name><name><surname>Larsen</surname><given-names>M. R.</given-names></name></person-group> (<year>2009</year>). <article-title>Analytical strategies for phosphoproteomics</article-title>. <source>Proteomics</source><volume>9</volume>, <fpage>1451</fpage>–<lpage>1468</lpage>. <pub-id pub-id-type="doi">10.1002/pmic.200800454</pub-id><pub-id pub-id-type="pmid">19235172</pub-id></mixed-citation></ref><ref id="B50"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Umezawa</surname><given-names>T.</given-names></name><name><surname>Sugiyama</surname><given-names>N.</given-names></name><name><surname>Takahashi</surname><given-names>F.</given-names></name><name><surname>Anderson</surname><given-names>J. C.</given-names></name><name><surname>Ishihama</surname><given-names>Y.</given-names></name><name><surname>Peck</surname><given-names>S. C.</given-names></name><etal></etal></person-group>. (<year>2013</year>). <article-title>Genetics and phosphoproteomics reveal a protein phosphorylation network in the abscisic acid signaling pathway in <italic>Arabidopsis thaliana</italic></article-title>. <source>Sci. Signal.</source><volume>6</volume>:<fpage>rs8</fpage>. <pub-id pub-id-type="doi">10.1126/scisignal.2003509</pub-id><pub-id pub-id-type="pmid">23572148</pub-id></mixed-citation></ref><ref id="B51"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Valero Galván</surname><given-names>J.</given-names></name><name><surname>Jorrín Novo</surname><given-names>J.</given-names></name><name><surname>Cabrera</surname><given-names>A.</given-names></name><name><surname>Ariza</surname><given-names>D.</given-names></name><name><surname>García-Olmo</surname><given-names>J.</given-names></name><name><surname>Cerrillo</surname><given-names>R.</given-names></name></person-group> (<year>2012a</year>). <article-title>Population variability based on the morphometry and chemical composition of the acorn in Holm oak (<italic>Quercus ilex</italic> subsp. ballota [Desf.] Samp.).</article-title><source>Eur. J. For. Res.</source><volume>131</volume>, <fpage>893</fpage>–<lpage>904</lpage>. <pub-id pub-id-type="doi">10.1007/s10342-011-0563-8</pub-id></mixed-citation></ref><ref id="B52"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Valero Galván</surname><given-names>J.</given-names></name><name><surname>Valledor</surname><given-names>L.</given-names></name><name><surname>Cerrillo</surname><given-names>R. M. N.</given-names></name><name><surname>Pelegrín</surname><given-names>E. G.</given-names></name><name><surname>Jorrín-Novo</surname><given-names>J. V.</given-names></name></person-group> (<year>2011</year>). <article-title>Studies of variability in Holm oak (<italic>Quercus ilex</italic> subsp. ballota [Desf.] Samp.) through acorn protein profile analysis.</article-title><source>J. Proteomics</source><volume>74</volume>, <fpage>1244</fpage>–<lpage>1255</lpage>. <pub-id pub-id-type="doi">10.1016/j.jprot.2011.05.003</pub-id><pub-id pub-id-type="pmid">21605712</pub-id></mixed-citation></ref><ref id="B53"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Valero Galván</surname><given-names>J.</given-names></name><name><surname>Valledor</surname><given-names>L.</given-names></name><name><surname>González Fernandez</surname><given-names>R.</given-names></name><name><surname>Navarro Cerrillo</surname><given-names>R. M.</given-names></name><name><surname>Jorrín-Novo</surname><given-names>J. V.</given-names></name></person-group> (<year>2012b</year>). <article-title>Proteomic analysis of Holm oak (<italic>Quercus ilex</italic> subsp. ballota [Desf.] Samp.) pollen.</article-title><source>J. Proteomics</source><volume>75</volume>, <fpage>2736</fpage>–<lpage>2744</lpage>. <pub-id pub-id-type="doi">10.1016/j.jprot.2012.03.035</pub-id><pub-id pub-id-type="pmid">22484522</pub-id></mixed-citation></ref><ref id="B54"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Valledor</surname><given-names>L.</given-names></name><name><surname>Jorrín</surname><given-names>J.</given-names></name></person-group> (<year>2011</year>). <article-title>Back to the basics: maximizing the information obtained by quantitative two dimensional gel electrophoresis analyses by an appropriate experimental design and statistical analyses</article-title>. <source>J. Proteomics</source><volume>74</volume>, <fpage>1</fpage>–<lpage>18</lpage>. <pub-id pub-id-type="doi">10.1016/j.jprot.2010.07.007</pub-id><pub-id pub-id-type="pmid">20656082</pub-id></mixed-citation></ref><ref id="B55"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Walters</surname><given-names>C.</given-names></name><name><surname>Berjak</surname><given-names>P.</given-names></name><name><surname>Pammenter</surname><given-names>N.</given-names></name><name><surname>Kennedy</surname><given-names>K.</given-names></name><name><surname>Raven</surname><given-names>P.</given-names></name></person-group> (<year>2013</year>). <article-title>Preservation of recalcitrant seeds</article-title>. <source>Science</source><volume>339</volume>, <fpage>915</fpage>–<lpage>916</lpage>. <pub-id pub-id-type="doi">10.1126/science.1230935</pub-id><pub-id pub-id-type="pmid">23430644</pub-id></mixed-citation></ref><ref id="B56"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Wang</surname><given-names>W.</given-names></name><name><surname>Vignani</surname><given-names>R.</given-names></name><name><surname>Scali</surname><given-names>M.</given-names></name><name><surname>Cresti</surname><given-names>M.</given-names></name></person-group> (<year>2006</year>). <article-title>A universal and rapid protocol for protein extraction from recalcitrant plant tissues for proteomic analysis</article-title>. <source>Electrophoresis</source><volume>27</volume>, <fpage>2782</fpage>–<lpage>2786</lpage>. <pub-id pub-id-type="doi">10.1002/elps.200500722</pub-id><pub-id pub-id-type="pmid">16732618</pub-id></mixed-citation></ref><ref id="B57"><mixed-citation publication-type="book"><person-group person-group-type="author"><name><surname>Woods Ignatoski</surname><given-names>K.</given-names></name></person-group> (<year>2001</year>). <article-title>Immunoprecipitation and western blotting of phosphotyrosine-containing proteins</article-title>, in <source>Protein Kinase Protocols</source>, ed <person-group person-group-type="editor"><name><surname>Reith</surname><given-names>A.</given-names></name></person-group> (<publisher-name>Humana Press</publisher-name>), <fpage>39</fpage>–<lpage>48</lpage>.</mixed-citation></ref><ref id="B58"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Wu</surname><given-names>R.</given-names></name><name><surname>Haas</surname><given-names>W.</given-names></name><name><surname>Dephoure</surname><given-names>N.</given-names></name><name><surname>Huttlin</surname><given-names>E. L.</given-names></name><name><surname>Zhai</surname><given-names>B.</given-names></name><name><surname>Sowa</surname><given-names>M. E.</given-names></name><etal></etal></person-group>. (<year>2011</year>). <article-title>A large-scale method to measure absolute protein phosphorylation stoichiometries</article-title>. <source>Nat. Methods</source><volume>8</volume>, <fpage>677</fpage>–<lpage>683</lpage>. <pub-id pub-id-type="doi">10.1038/nmeth.1636</pub-id><pub-id pub-id-type="pmid">21725298</pub-id></mixed-citation></ref></ref-list></back></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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