The document summarizes a study that examined the effects of different concentrations of tropospheric ozone exposure on soybean plants through proteomic analysis. It found that ozone exposure significantly altered the expression and oxidation states of proteins involved in important metabolic pathways like carbon metabolism and nitrogen homeostasis. In particular, proteins related to photosynthesis like RuBisCO showed increased oxidation and expression with higher ozone levels, which could reduce their catalytic activity and photosynthetic capacity.

1. Joseph Jez
Presentation by : Satya Prakash Chaurasia
Department of Botany,
University of Delhi,
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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.