Towards the identification of late-embryogenic-abundant phosphoproteome in Arabidopsis by 2-DE and MS.
Late-embryogenesis-abundant (LEA) proteins accumulate as plant seeds desiccate and also in vegetative organs during periods of stress. They are predicted to play a role in plant stress tolerance. In the present study, we have initiated the characterization of phosphorylated LEA proteins present in the Arabidopsis seed, using a strategy that combines the thermostability (solubility upon heating) of many LEA-type proteins with the use of phosphoaffinity chromatography to obtain an enriched subpopulation of phosphoproteins. The specificity and efficiency of the procedure was assessed by alkaline phosphatase treatment and by a specific stain for phosphoproteins, in addition to the immunodetection of AtRab18, a phosphorylated LEA protein present in the mature dry seed. The phosphoproteins were identified by MS either by PMF using MALDI-TOF MS after 2-DE separation, or by peptide sequencing using both capillary LC MS/MS (LC muESI-ITMS/MS) and nanoLC coupled to nanoESI-MS/MS (LC-nanoESI-Q-TOF-MS/MS). Several LEA-type and storage-like proteins were identified as components of the phosphoproteome of the Arabidopsis seed.
Irar
Sami
S
Laboratori de Genètica Molecular Vegetal, Institut de Biología Molecular de Barcelona, Consorci CSIC-IRTA, Barcelona, Spain.
Oliveira
Eliandre
E
Pagès
Montserrat
M
Goday
Adela
A
D017360:Arabidopsis / Q000196:embryology / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D018506:Gene Expression Regulation, Plant / Q000502:physiology; D010750:Phosphoproteins / Q000378:metabolism*; D020543:Proteome / Q000378:metabolism*; D012639:Seeds / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D020691:rab GTP-Binding Proteins / Q000378:metabolism
D029681:Arabidopsis Proteins; D010750:Phosphoproteins; D020543:Proteome; C078703:rab18 protein, Arabidopsis; D020691:rab GTP-Binding Proteins