Abstract
Hydrogen peroxide (H2O2) plays a dual role in plants as the toxic by-product of normal cell metabolism and as a regulatory molecule in stress perception and signal transduction. However, a clear inventory as to how this dual function is regulated in plants is far from complete. In particular, how plants maintain survival under oxidative stress via adjustments of the intercellular metabolic network and antioxidative system is largely unknown. To investigate the responses of rice seedlings to H2O2 stress, changes in protein expression were analyzed using a comparative proteomics approach. Treatments with different concentrations of H2O2 for 6 h on 12-day-old rice seedlings resulted in several stressful phenotypes such as rolling leaves, decreased photosynthetic and photorespiratory rates, and elevated H2O2 accumulation. Analysis of approximately 2000 protein spots on each two-dimensional electrophoresis gel revealed 144 differentially expressed proteins. Of them, 65 protein spots were up-regulated, and 79 were down-regulated under at least one of the H2O2 treatment concentrations. Furthermore 129 differentially expressed protein spots were identified by mass spectrometry to match 89 diverse protein species. These identified proteins are involved in different cellular responses and metabolic processes with obvious functional tendencies toward cell defense, redox homeostasis, signal transduction, protein synthesis and degradation, photosynthesis and photorespiration, and carbohydrate/energy metabolism, indicating a good correlation between oxidative stress-responsive proteins and leaf physiological changes. The abundance changes of these proteins, together with their putative functions and participation in physiological reactions, produce an oxidative stress-responsive network at the protein level in H2O2-treated rice seedling leaves. Such a protein network allows us to further understand the possible management strategy of cellular activities occurring in the H2O2-treated rice seedling leaves and provides new insights into oxidative stress responses in plants.
Highlights
Hydrogen peroxide (H2O2) plays a dual role in plants as the toxic by-product of normal cell metabolism and as a regulatory molecule in stress perception and signal transduction
Similar patterns of rate decrease were observed for the stomatal conductance (Gs), the intercellular CO2 concentration (Ci), and the transpiration speed (Ts) (Fig. 1C), suggesting that H2O2 treatment resulted in stomatal closure, further reduced water transpiration, and exoteric CO2 absorption in the treated rice seedling leaves
The present proteomics study revealed 144 H2O2-responsive spots on the 2-DE gel image containing about 2000 reproducible spots from which 129 differentially expressed proteins were successfully identified in H2O2treated rice seedling leaves
Summary
Chemicals—CHAPS, IPG DryStrip, IPG buffer, and iodoacetamide were purchased from GE Healthcare; thiourea and n-octyl glucopyranoside were from Sigma; and trypsin (MS Gold), urea, and acrylamide were from Promega (Madison, WI). The middle portions of the second and third leaves were collected and frozen in liquid nitrogen and stored at Ϫ80 °C for protein extraction. Rice seedling leaves (1.0 g) were ground with a mortar and pestle in liquid nitrogen to a fine powder and added to a 10-ml cuvette containing 8 ml of double distilled H2O and 2 ml of 25 mM titanium sulfate and incubated for 1 h at room temperature. The protein extract was diluted to a final concentration of 2500 g/ml with an IEF rehydration solution (2% (w/v) CHAPS, 0.5% IPG buffer, 2 M thiourea, and 6 M urea). In-gel digestion of protein spots was performed according to Yao et al (22). The theoretical values of molecular weight (Mr) and pI of identified proteins were predicted by using the PeptideMass program (ExPASy)
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