1. Analysis of response mechanism in soybean under low oxygen and flooding stresses using gel-base proteomics technique
Amana Khatoon• Myeong-Won Oh•Sun-Hee Woo• Setsuko Komatsu
National Institute of Crop Science, National Agriculture and Food Research Organization,Japan
Department of Plant Sciences, Kohat University of Science and Technology
Mol Biol Rep (2012) 39:10581–10594

2. Background
Low oxygen conditions lead to loss of plasma membrane integrity that membrane integrity is the key factor responsible for plant survival under hypoxic conditions .
Flooding stress imposed hypoxic conditions to plants.
hypoxic stress affected the amount of many gene transcripts, and that many accumulated mRNAs were not translated into proteins during oxygen deprivation.
Soybean is sensitive to flooding, which caused a decrease
in photosynthesis , nutrient uptake and nitrogen fixation

3. In this study
Clarify the plant response towards low oxygen and flooding stresses at early growth stage of soybean seedlings
Under these stresses will provide a key to make strategies for low oxygen and flood tolerant soybean.

4. methods 2-DE MALDI-TOF-MS Nano-LC MS/MS Student’s t test
One-way ANOVA Duncan’s multiple comparisons test

5. Materials and methods Plant growth conditions
Soybean seeds were sterilized with sodium hypochlorite solution, rinsed in water and were germinated on sand under white fluorescent light
Three-day-old soybean seedlings were imposed to flooding and low oxygen stress separately for 6 days.
Physiological parameters including lengths and fresh weights of root, shoot and number of secondary roots were measured on 3 and 6 days following the treatments. For the analysis of differential changes in proteins, roots were collected on 3 days after treatment.
All experiments were biologically repeated three times with 30 and 16 seeds per replication for physiological and proteomics experiments, respectively
600umolm- 2s- 1, 12 h light period day- 1. at 25 °C and 70 % relative humidity
Low oxygen ：substituting the
normal air by nitrogen supply in the closed container.
Flooding ：adding water to
complete submergence of germinating seedlings

6. Protein extraction supernatant was discarded
and resulting pellet was wa shed with 0.07 % 2-mercap-toethanol in acetone twice.
be ground to powder in
liquid nitrogen
sonicated and
incubated
centrifuged
10 % trichloroacetic acid and 0.07 % 2-mercap-toethanol in acetone
9,000g for 20 min at 4°C.
sonicated for 5 min and then incubated for 1 h at -20°C
Dried：Speed-Vac concentrator
Resuspended：lysis buffer 1 h at 25°C
Supernatant was collected as
protein extract. Protein contents were determined using the Bradford method with bovine serum albumin as the standard.
dried
and
resuspended

7. 2-DE
Protein extract in a final volume of 200 uL of lysis were directly loaded into a focusing tray. The immobilized pH gradient strips (3–10NL, 11 cm, Bio-Rad) were rehydrated for 14 h at 50 V.
Sample preparation
250 V for 15 min with a linear ramp, 8,000 V for 1 h with a linear ramp, and finally 8,000 V at 35,000 V h- 1 with a rapid ramp at 20°C
IEF
6 M urea, 2 % SDS, M Tris–HCl (pH 8.8), 20 % glycerol, and 130 mM dithio-threitol for 30 min.
6 M urea, 2 % SDS, M Tris–HCl (pH 8.8), 20 %glycerol, and 135 mM iodoacetamide for 30 min.
Equilibration twice
15 % SDS–polyacryl-amide gels with 5 % stacking gels and sealed with 1 %
agarose. Electrophoresis in the second dimension was per-formed at a constant current of 35 mA.
SDS-PAGE
CBB
PDQuest
Gel image analysis

8. Protein identification
By MALDI-TOF MS
These are as follows:
(i) the deviation between the experimental and the theoretical peptide masses should be less than 50 ppm.
(ii) At least 5 different predicted peptide masses needed to match the observed masses for an identi-fication to be considered valid.
(iii) The coverage of protein sequences by the matching peptides must reach a minimum of 16 %.
(iv) The score indicates the probability of a true positive identification and it must be at least 60.

9. By nano-LC MS/MS These are as follows:
(i) The score indicates the probability of a true positive identification and it must be at least 100.
(ii) At least 3 peptide sequence matches above the identity threshold.
(iii) The coverage of protein sequences by the matching peptides must reach a minimum of 9 %.

10. Results
Changes in plant growth under low oxygen and flooding stresses

12. Differential change of proteins in seedlings under low oxygen and flooding stresses

15. Differential change of common proteins between low oxygen and flooding stresses

18. Identification of increased and decreased proteins in soybean seedlings under low oxygen and flooding stresses

19. Discussion
In this study growth suppression occurred under low oxygen and flooding stresses with more severe reduction in flooded seedlings (Fig. 1 ).
This difference in number and intensity level of proteins indicated far complex nature of flooding stress than being interpreted only in terms of oxygen deprivation. (Fig. 3,4 ).

20. Kunitz trypsin protease inhibitor was remarkably increased in flooded soybean seedlings.
The increase in this enzyme in flooded seedlings compared to low oxygen treated seedlings indicated that suppression of growth was more severe in flooded seedlings and root damage due to physical injuries under flooding stress caused greater growth suppression compared to low oxygen stress (Fig.1 )

21. Alcohol dehydrogenase was remarkably increased in low oxygen treated seedlings compared to increase in flooded soybean seedlings (Fig. 3 ).
indicated that fermentation pathway is the main adaptation of plants due to low oxygen, acting as prime signal in flooded seedlings to adapt to fermentation pathway (Fig. 3 )

22. UDP-glucose 6-dehydrogenase was high in flooded seedlings.
indicated the more efficient modification of cell structure and regulation of energy generation to cope with more severe energy crises imposed by flooding.

23. D-3-Phosphoglycerate dehydrogenase and pyruvate decarboxylase were also increased under low oxygen and flooding stresses with more degree of increase of under flooding stress.
3-phosphoglycerate dehydrogenase might help soybean seedlings to synthesize amino acids that could be a stress responsive strategy.
pyruvate decarboxylase play a critical role in maintaining the energy production under the flooded and low oxygen conditions.

24. Among the proteins whose abundance was decreased commonly under low oxygen and flooding stresses, the decrease in quinone oxidoreductase and chalcone isomerase A was more in flooded seedlings.
The more decrease of quinone oxidoreductase in flooded seedlings thus present the more growth suppression in flooded soybean seedlings.
The decrease in chalcone isomerase presents decreased activity of this enzyme in flavonoid biosynthesis which might lead to decreased control over primary leaf organogenesis.

25. Among the proteins which specifically changed in seedlings under low oxygen stress, TCP domain class transcription factor was decreased compared to control.
might affect the cell division in root meristem thus leading to decreased cell proliferation and growth in low oxygen stressed seedlings

26. Among the flooding specific proteins, NADP-dependent malic enzyme were decreased.
The decrease of NADP-dependent malic enzyme in flooded seedlings might lead to susceptibility to diseases which suppressed the growth under flooding stress.

27. in short
soybean seedlings subjected to flooding stress are affected by not only hypoxic stress but multiple stresses such as mechanical injuries due to water, and stresses imposed by cold and weak light under complete submergence.
Response of soybean to low oxygen and flooding stresses is different at protein level also with difference in number and pattern of protein changes
Physiological and proteomics results suggest that changes in flooded seedlings were more drastic compared to low oxygen stressed seedlings, which lead to plant injuries and resulted in growth suppression.

28. Thanks!