A comparative proteomic analysis of tomato leaves in response to waterlogging stress

Abstract

A comparative proteomic approach has been adopted in combination with physiological and biochemical analysis of tomato leaves responding to waterlogging stress. Waterlogging resulted in increases of relative ion leakage, lipid peroxidation and in vivo H2O2 content, whereas the chlorophyll content was decreased. Histocytochemical investigations with 3,3'-diaminobenzidine to localize H2O2 and Evans blue to detect dead cells suggested that oxidative stress has a significant role to leaf senescence. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), the most abundant leaf protein, was successfully reduced from the samples by a fractionation method based on 15% polyethylene glycol (PEG). Elimination of Rubisco was further confirmed by Western blot analysis. To elucidate the temporal changes of the protein patterns in tomato leaves, the total soluble and the PEG-fractionated proteins were separated by two-dimensional electrophoresis (2-DE) and visualized by Coomassie Brilliant Blue staining. A total of 52 protein spots were differentially expressed, wherein 33 spots were identified by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry or electrospray ionization tandem mass spectrometry (ESI-MS/MS) analysis. The identified proteins are involved in several processes, i.e. photosynthesis, disease resistance, stress and defense mechanisms, energy and metabolism and protein biosynthesis. Results from 2-DE analysis, combined with immunoblotting clearly showed that the fragments of Rubisco large subunit were significantly degraded. This could result from a higher production of reactive oxygen species in leaves under waterlogging stress. Furthermore, four differentially accumulated proteins were analyzed at the mRNA level, confirming the differential gene expression levels and revealing that transcription levels are not always concomitant to the translation level. A number of novel proteins were differentially expressed or appeared only in the PEG-fractionated protein samples, indicating that PEG fractionation system can be used as a versatile protein fractionation technique in proteomic analysis to identify novel or low-abundant proteins from all kinds of plant species.

Proteomic Analysis of Lettuce Seed Germination and Thermoinhibition by Sampling of Individual Seeds at Germination and Removal of Storage Proteins by Polyethylene Glycol Fractionation

“…This situation occurs also in other plant tissues, such as leaf, in which Rubisco is highly abundant Acquadro et al, 2009). PEG fractionation is recognized as a convenient and reproducible method for the removal of Rubisco and to increase the resolution of low-abundance proteins in leaf extracts of many species (Xi et al, 2006;Ahsan et al, 2007;Lee et al, 2007;Acquadro et al, 2009). This method is clearly also applicable to seed samples ( Fig.…”

“…PEG is an excellent general reagent for differential precipitation of proteins, and PEG fractionation is simpler and more effective in removing high-abundance proteins (Juckes, 1971). This approach has been applied to effectively remove most of the Rubisco in globe artichoke (Cynara scolymus; Acquadro et al, 2009), tomato (Solanum lycopersicum; Ahsan et al, 2007), rice , and Arabidopsis (Xi et al, 2006) leaves. In addition, it increases the number of detectable protein spots and allows for the detection of many low-abundance proteins in these tissues.…”

The first assessment of the stress inducible defense of Leucaena leucocephala with acaricidal potential effect against Rhipicephalus (Boophilus) microplus (Acari: Ixodidae)

“…The defense mechanism is orchestrated by changes in protein expression, in which the plant is able to overexpress or inhibit the expressions or actions of molecules such as protease, protease inhibitor, catalase, peroxidase, chitinase and lipoxygenase (WANG et al, 2003;AHSAN et al, 2007;GULSEN et al, 2010;KOSOVÁ et al, 2011).…”

Expression of Heat Shock Protein and Antioxidant Genes in Rice Leaf Under Heat Stress

“…4). Up-regulation of glyoxalase I at the proteome level has also been monitored in plants exposed to several abiotic stresses (Khan et al, 2005;Ahsan et al, 2007). Based on these observations, we propose that glyoxalase I may play a pivotal role in detoxifying the toxic substances induced by thermal stress in plants.…”

“…The H2O2 concentrations of treated and control leaves were measured spectrophotometrically, as described previously (Ahsan et al, 2007). Proline content was determined according to the method of Bates et al (1973).…”