Investigating the Impact of Elevated Levels of Ozone on Tropical Wheat Using Integrated Phenotypical, Physiological, Biochemical, and Proteomics Approaches

Abstract

Ozone (O3), a potent air pollutant and a significant greenhouse gas, has been recognized as a major component of global climate change. However, current increasing trends in its background level are projecting a more severe threat to natural and cultivated plants in the near future. The present study has been designed to evaluate the impact of elevated concentrations of O3 on phenotypical, physiological, and biochemical traits in two high-yielding cultivars of wheat, followed by analysis of the leaf proteome using one/two-dimensional gel electrophoresis (1-/2-DGE) coupled to immunoblotting and mass spectrometry analyses under near-natural conditions using open top chambers. Prominently, O3 exposure caused specific foliar injury in both the wheat cultivars. Results also showed that O3 stress significantly decreased photosynthetic rate, stomatal conductance, and chlorophyll fluorescence kinetics (Fv/Fm) in test cultivars. Biochemical evaluations further revealed a higher loss in photosynthetic pigments, whereas a significantly induced antioxidant system under elevated O3 concentrations pointed toward an ability of O3 to generate oxidative stress. 1-DGE analysis showed drastic reductions in the abundantly present ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) large and small subunits. Western blot analysis confirmed induced accumulation of antioxidative enzymes like superoxide dismutase and ascorbate peroxidase protein(s) and common defense/stress-related thaumatin-like protein(s). 2-DGE analysis revealed a total of 38 differentially expressed protein spots, common in both the wheat cultivars. Among those, some major leaf photosynthetic proteins (including RuBisCO and RuBisCO activase) and important energy metabolism proteins (including ATP synthase, aldolase, and phosphoglycerate kinase) were drastically reduced, whereas some stress/defense-related proteins (such as harpin-binding protein and germin-like protein) were induced. In all, the present study reveals an intimate molecular network provoked by O3 affecting photosynthesis and triggering antioxidative defense and stress-related proteins culminating in accelerated foliar injury in wheat plants.

From climate change to molecular response: redox proteomics of ozone‐induced responses in soybean

“…3a). Overall, the changes in protein abundance observed in the high-O 3 root and the moderate-O 3 leaf and root samples were comparable to those described in previous studies of plant proteomes following short-term O 3 exposure in growth chambers (Agrawal et al, 2002;Bohler et al, 2007;Cho et al, 2008;Feng et al, 2008;Renaut et al, 2009;Ahsan et al, 2010;Sarkar et al, 2010).…”

“…Likewise, decreased photosynthetic efficiency, reduced RuBisCO activity, and elevated PEPC activity are classic markers for O 3 damage and senescence; however, these effects can vary with the length, concentration, and type of exposure (Heagle, 1989;Chernikova et al, 2000;Robinson & Britz, 2000;Krupa et al, 2001;Morgan et al, 2004Morgan et al, , 2006Baier et al, 2005;Fiscus et al, 2005;Chen et al, 2009;Betzelberger et al, 2010). Previous studies on soybean, rice, and wheat exposed to constant 120-200 ppb O 3 for 3-5 d in growth chambers typically led to the identification of 20-50 proteins that changed in abundance either up or down (Agrawal et al, 2002;Bohler et al, 2007;Cho et al, 2008;Feng et al, 2008;Renaut et al, 2009;Ahsan et al, 2010;Sarkar et al, 2010). The actual proteins identified in these short-term O 3 exposure experiments vary widely, but decreased abundance of the RuBisCO large and small subunits and increased ascorbate peroxidase concentrations are generally observed.…”

“…Elevated O 3 decreases photosynthetic rates and both seed mass and number (Chernikova et al, 2000;Robinson & Britz, 2000;Krupa et al, 2001;Baier et al, 2005;Fiscus et al, 2005;Morgan et al, 2006;Chen et al, 2009;Emberson et al, 2009;Betzelberger et al, 2010). At the molecular level, proteomic studies of rice, wheat, soybean, and poplar exposed to continuous short-term (2-5 d) high (> 200 ppb) O 3 stress in growth chambers generally reveal drastic reductions in the major leaf photosynthetic and carbon metabolism proteins and induction of defense ⁄ stress-related proteins (Agrawal et al, 2002;Bohler et al, 2007;Cho et al, 2008;Feng et al, 2008;Renaut et al, 2009;Ahsan et al, 2010;Sarkar et al, 2010); however, assessments of proteome changes have not examined plants grown in the field under long-term elevated daytime O 3 concentrations. Moreover, published studies do not probe the effect of O 3 on the plant thiol-redox proteome, as these changes are not observable by standard proteomics methods (Buchanan & Balmer, 2005;Alvarez et al, 2011).…”

Ozone Exposure Response for U.S. Soybean Cultivars: Linear Reductions in Photosynthetic Potential, Biomass, and Yield

“…However, chronic or long-term exposure of plants in the field to lower [O 3 ] does not necessarily elicit the same transcriptional or metabolite response as short-term acute O 3 exposure (Gillespie et al, 2012). At lower concentrations, O 3 may not cause visible necrotic lesions on leaves but can negatively impact photosynthetic carbon gain via effects on the Calvin cycle and light-harvesting processes (Goumenaki et al, 2010;Sarkar et al, 2010) and by accelerating senescence (Pell et al, 1997). Exposure to elevated [O 3 ] also fundamentally alters other aspects of metabolism, including increased demand for respiratory energy and increased flux through the shikimate, phenylpropanoid, and anapleurotic pathways (Dizengremel et al, 2009(Dizengremel et al, , 2012.…”

Plant proteomics in India and Nepal: current status and challenges ahead

“…Ozone stress Effect of elevated ozone (O 3 ) was studied on two high-yielding cultivars of tropical wheat (Sarkar et al 2010). Exposure to elevated O 3 resulted in foliar injury in both the cultivars (Sonalika and HUW 510).…”