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Ozone effects on plants...

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

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

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  • Soyabean being sensitive to oxidant activity of ozone was taken …. Highly sensitive to ozone.
  • In the northern hemisphere tropospheric ozone is the major air pollutant that causes yield loss in many important crops for e.g rice , wheat, soyabean etc. It enters leaves through stomata and produces ROS, induces premature leaf seniscence and imposes many other nagetive physiological effects ultimately adding to reduced phtosyntheticeffecieny and yield loss. Understanding how crops respond to increasing O3 pollution is essential for meeting the growing demands for sustainable food systems. Chlorophyll loss , leaf bronzing, necrotic spots. Cumulating into decreased photosynthetic effeciency. Economically, annual crop losses due to O3 damage at current tropospheric values are estimated at $2–4 billion in the US and $3–5.5 billion in China, and will likely increase in the future (Van Dingenen et al., 2009). Poplar. Reduction in proteins involved in photosy. And carbon metabolism and rise in defence/stress related proteins.
  • Soyabeans were exposed to ozone 9h per day. Either there is no apecific reason i.e they just waited in order to give plants more long exposure or they wanted to confirm whether there are effects on reproductive growth also. Rates of mature leaves from the upper canopy.
  • Between the moderate- and high-O3concentrations, there is a clear shift toward increased expressionand protein sulfhydryl oxidation across metabolism in the leaftissue of soybean.AnalysisThis suggests a thresholdbetween 58 and 116 ppb O3 at which the expression andoxidation of multiple proteins in leaf tissue drastically increase. data indicating that responses to constantshort-term vs seasonal daytime O3 exposure and to exposure atmoderate and high O3 differ. All of the proteins above interact with thioredoxin, which isessential for maintaining protein redox-state in plants. It is possible that these changes reflect the needto maintain MDH in the leaf to supply metabolites to the citricacid cycle.
  • Overall, the changes in protein abundance observed in the high-O3 root and the moderate-O3 leaf and root samples were comparable to those described in previous studies of plant proteomes following short-term O3 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).
  • Comparison between the tissue–O3 treatment combinations revealed increased abundance and greater oxidation of the largest number of proteins in the high-O3 leaf sample.
  • volatile isoprenoid emissions may actas an O3 protection mechanism in plants…. Carotenoid these compounds, which act as photoprotectivecompounds and antioxidants
  • All of the proteins above interact with thioredoxin, which is essential for maintaining protein redox-state in plants ( chloro– may help maintain photosynthesis)
  • If we compare the changes in total proteome of tissue b/w short term and long term exposure studies , the results differ for e.g decreased abundance of Rubi large nad small subunit while in long term exposure the subunits are increased in abundance.

Transcript

  • 1. Joseph Jez Presentation by : Satya Prakash Chaurasia Department of Botany, University of Delhi, satya.prakash101991@gmail.com www.facebook.com/91satya www.twitter.com/Satyapr101991 h ttp://www.linkedin.com/pub/satya-prakash chaurasia/65/a76/a25 Mob: +919654814497
  • 2. Introduction  Tropospheric ozone in northern hemisphere causes significant Agricultural loss.  Since 90th century, ground level O3 concentrations have doubled.  Regions in India , china and US facing a 10% per year rise.  Soybean, rice , wheat etc.  Climate models predict that mean surface O3 concentrations may rise 20– 25% globally by 2050, with concentrations in India and south Asia reaching comparable values by 2020 (IPCC 2001; Dentener et al., 2005; Van Dingenen et al., 2009).
  • 3. Basic Protocol  O3 treatment. Soybeans were planted at SoyFACE in July, 2009 three different treatments were given[Ambient(36ppb),moderate(58),high(116ppb)] plants were harvested during reproductive growth in Aug ,2009 leaves and roots were obtained and immediately dipped in liquid N2 • Bradford Assay.Total Protein • Infrared gas analyzerPhotosynthetic carbon uptake rates
  • 4.  Protein extraction/redox proteome labeling Frozen tissues were ground to a fine powder in liquid N2 suspended in extraction buffer( containing NEM) Sonication and centrifuged supernatant mixed with MeOH,incubation centrifuged pellet washed with MeOH and resuspended in reduction buffer( DDT) Incubation and reprecipitation with MeOH Pellet dried and resuspended in labeling buffer( IAF) NEM: N-ethylmaleimide ; IAF: 5-iodoacetamidofluorescein
  • 5.  IEF run and proteomic analysis proteins absorbed into IEF pH(4-7) strips imaging of gel to detect IAF labeled proteins gels were fixed, washed and stained with Sypro Ruby to detect total protein image alignment and identification of spots differing significantly these spots were excised and proteins obtained LC- MS/MS analysis and Data searched against NCBInr database
  • 6.  Spectrophotometric Enzyme assays: • Phosphoglycerate Kinase(PGK) • Malate dehydrogenase(MDH) • Glutamine synthase(GS) • Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) • Fructose 1,6-bisphosphate aldolase ( FBA), and • Ribulose 1,5-bisphosphate carboxylase oxygenase ( RuBisCo)  Immunoblot analysis of RuBisCo large subunit: membrane from SDS-PAGE, after TBS incubation, treated with Anti- RuBisCo large subunit antibody treated with secondary antibody conjugated with enzyme detection by colorimetric detection
  • 7. Results • Total protein content from different plant samples showed no significant difference. • Photosynthetic carbon uptake showed linear decline with increasing Ozone concentration. • A total of 1455 spots were identified of which 277 were differentially expressed and/or oxidized. • Effects vary with length, concentration of Ozone exposure.
  • 8. (a) Distribution of proteins across combinations of tissue and O3 concentration. Numbers in overlapping regions of the lobes indicate proteins found in more than one set of conditions. (b) (b–e) Numbers of differentially oxidized (5- iodoacetamidofluor escein (IAF)) and ⁄ or abundant (Sypro Ruby) proteins between treated samples and controls.
  • 9. Panels show the fold changes in oxidation (5-iodoacetamidofluorescein (IAF)) and expression level (Sypro) relative to ambient controls for proteins identified by nano-LC ⁄MS⁄ MS. Summary of fold-changes in total and redox proteomes of soybean root tissue exposed to high (116 ppb) O3 (a); and leaf (b) and root tissues (c) exposed to moderate (58 ppb) O3.
  • 10. Fold changes, relative to ambient (37 ppb) O3 sample, in oxidation state (5- iodoacetamidofluorescein (IAF)) and abundance (Sypro) for identified proteins are plotted. Names of representative proteins are shown with highly (> threefold) oxidized (orange box) and oxidized (> threefold) ⁄ expressed (> 1.5-fold) proteins (pink boxes) indicated. Summary of fold changes in total and redox proteomes of soybean leaf tissue exposed to high (116 ppb) O3.
  • 11. Discussion • Large no. of metabolic pathways are affected: – Amino acid biosynthesis – Nitrogen homeostasis – Carbon metabolism – Starch/sugar mobilization pathway(MDH) – Isoprenoid synthesis pathway – Carotenoid and isoflavanoids pathways – Calvin cycle , glycolysis, citric acid cycle
  • 12. • Glutamine synthase(GS) -leaf senescence and recycling of ammonia. • Malate dehydrogenase(MDH) Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and Fructose 1,6-bisphosphate aldolase ( FBA) • chlorophyll α ⁄ β binding protein, ferredoxin reductase, and a chlorophyllase-like protein RuBisCO large and small subunits, RuBisCO activase, RuBisCO-associated protein, and RuBisCO-binding protein showed increased expression and ⁄ or oxidation in this sample. Oxidation of RuBisCO can reduce the catalytic activity of the enzyme (Marcus et al., 2003); however, alterations in proteins associated with RuBisCO (i.e. the activase) may compensate for possible oxidative changes.
  • 13. The proteomic analysis here supports studies demonstrating that redox- protection mechanisms play a critical role in plant responses to O3 exposure (Gillespie et al., 2011), and for the first time directly shows that O3 exposure changes the thiol oxidation state of proteins in various pathways.