workshop on interdisciplinary plant science - … · marco a. z. arruda cp4 epsps...
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Marco Aurélio Zezzi ArrudaFull Professor
http://gepam.iqm.unicamp.br
http://www.inctbio.iqm.unicamp.br
e-mail: [email protected]
Workshop on Interdisciplinary Plant Science
Marco A. Z. Arruda
cp4 EPSPS (5-enolpyruvylshikimate-3-
phosphate synthase) gene from the soil
bacteria Agrobacterium sp. line cp4;
Genetic modification
The transgenic
Results
Seeds
Marco A. Z. Arruda
Transgenic soybean: Roundup Ready
Varieties:
MSOY 7575 RR (transgenic – T)
MSOY 7501 (non transgenic – NT)
Marco A. Z. Arruda
Proteomic profile: 4-7 pH range
192 identified proteins
Identification of the CP4 EPSPS – involved in the genetic modification
Marco A. Z. Arruda
Actin (fragment) Cytosolic glutamine
synthetase
Glycinin subunit G1
Glycine-rich
RNA-binding protein
-Cellular motility
-Bind ATP
and others proteins
Proteomic profile: 2-D DIGE
-Involved in the cellular
response to the environment
and development conditions
-Expressed in roots
-Binding to ATP
-Fixation of N
-Biosynthesis of glutamine
-Nutritional,
physical-chemical,
physiological properties
of seed
Soybean seeds APX1 CAT2 GR3 SOD4
T 18.84±0.003 3.37±0.10 0.14±0.01 42.6±1.81
NT 13.08±0.002 2.25±0.02 0.04±0.01 81.5±3.11
T/NT 1.44 1.50 3.5 0.52
Marco A. Z. ArrudaEnzymatic activity
1 mM ascorbate.min-1 mg protein-1; 2 nM H2O2.min-1 mg protein-1;3 μM GSSH.min-1 mg protein-1; 4units of SOD.mg protein-1
Lipid peroxidationMDA (nM/g):
T 2.25±0.04
NT 1.56±0.06
Seeds present a considerable stress!!
Marco A. Z. Arruda
200 mg seedsVarieties
MSOY 7501 and MSOY7575RR
Microwave assisted decomposition
(35 min)
6.3 mL HNO3 + 0.75 mL H2O2
Dry to constant
mass
Total determination
Marco A. Z. Arruda
Soybean seeds, µg g-1
T NT
Al 0.08±0.04 <0.24a
As <0.0009a <0.0009a
Ba 0.104±0.004 <0.002b
Bi <0.001a <0.001a
Ca 935±37 859±36
Cd <0.0006b <0.0006b
Co 0.045±0.001 0.028±0.001
Cr <0.15a <0.15a
Cu 10.3±0.3 6.2±0.8
Fe 81.2±2.3 65.0±2.4
Hg <0.0015a <0.0015a
Li <0.006a <0.006a
Mg 3684±52 3468±507
Mn 28.1±0.6 25.1±2.3
Mo 0.31±0.03 0.34±0.06
Ni 0.49±0.08 0.32±0.08
P 6645±377 6573±718
Pb 0.006±0.004 0.009±0.003
S 3006±97 3208±242
Sb 0.001±0.001 0.001±0.001
Se 0.11±0.04 <0.02b
Sn <0.031a <0.031a
Sr 21.2±0.6 30.5±3.3
Te <0.0003a <0.0003a
Zn 41.3±1.1 39.5±1.7
a LODb LOQ
Total
determination
Mataveli et al., Metallomics, 2(2010)800
Marco A. Z. Arruda*Mounicou, S., et al., J. Anal. At. Spectrom., 17(2002)880
0.3 g seeds + 5 mL gastric solution
(pepsine + NaCl)
Incubation 37°C @ 4h
10 mL de intestinal solution
(pancreatin + amylase + biliary salts)
Centrifugation
(ICP-SF-MS)
Incubation 37°C @ 4h
Bioaccessibility test*
Marco A. Z. Arruda
Concentration, µg g-1Recovery %
Bioaccessible fraction Residues
T NT T NT T NT
Ca 824±114 817±72 160±17 193±8 105±13.2 117±12.7
Cu 3.52±0.13 1.85±0.02 4.77±0.57 3.39±0.20 80±8.1 85±8.6
Fe 29.1±2.4 5.99±0.26 52.6±3.8 59.0±9.1 101±6.3 100±6.8
Mg 1690±248 1318±78 492±53 511±20 91±11.9 75±7.6
Mn 12.2±0.1 10.5±0.3 7.67±0.90 7.77±0.33 71±9.0 73±10.0
S 1322±56 1961±76 958±64 1476±68 73±8.8 107±9.6
Zn 22.1±0.1 19.6±0.4 15.7±0.6 22.6±0.8 92±2.3 107±7.6
Ca, Cu, Fe, Mg, Mn, S and Zn concentrations in
bioaccessible fractions, residues and recoveries of
transgenic (T) and non-transgenic (NT) soybean seeds
(average values, n=3 ± standard deviation)
Soybean cultivation
Marco A. Z. Arruda
Plants cultivated in BOD chamber 27±0.1oC
Photoperiod: 12h
Substrate for plants (BasaPlanta+Vermflock agro)
Water supply in alternate days
Germination: ca. 3 days
Leaves were harvest at 10th (T1) and 15th (T2) days
of cultivation (but not from the same plant)
Leaves APX CAT GR SOD
T1 546±0.002 105±0.0006 0.154±0,006 7.7±0.036
T2 429±0.001 94.1±0.001 0.148±0,004 5.5±0.027
NT1 431±0.002 90.5±0.0012 0.046±0,003 5.3±0.020
NT2 414±0.002 69.7±0.0015 0.037±0.001 5.1±0.028
T1/NT1 1.27 (21%) 1.16 (14%) 3.35 (70%) 1.45 (31%)
T2/NT2 1.04 (3.5%) 1.35 (26%) 4.00 (75%) 1.08 (7.3%)
Enzymatic activity
T1 – 10th day
T2 – 15th day
Marco A. Z. Arruda
1 mM ascorbate.min-1 mg protein-1;2 nM H2O2.min-1 mg protein-1;3 μM GSSH.min-1 mg protein-1;4units of SOD.mg protein-1
Hydrogen peroxide
Marco A. Z. Arruda
0
0,005
0,01
0,015
0,02
0,025
NT 1 NT 2 T1 T2
Hyd
rog
en
pe
rox
ide
(m
mo
l/L
)
0.0131
±0.0009
0.008
±0.0003
0.022
±0.0018
0.0091
±0.0005
Lipid peroxidation
0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
NT 1 NT 2 T1 T2
MD
A
(µM
/g f
res
hm
ate
ria
l)
0.4798
±0.005
0.4651
±0.016
0.6806
±0.001
0.6951
±0.001
Leaves present considerable stress!!
NT1xT1NT1 T1 NT1 T1
NT1 T1
NT1 T1
NT1 T1
NT1 T1
• 47 differential spots:
• 19 more abundant (blue)
• 28 less abundant (red)
Proteomic profile: 2-D DIGE
Marco A. Z. Arruda
NT2 T2
NT2 T2
NT2 T2
NT2 T2
NT2 T2
NT2 T2
• 28 differential spots:
• 25 more abundant (blue)
• 3 less abundant (red)
Proteomic profile: 2-D DIGE
NT2xT2
Marco A. Z. Arruda
Differential protein species
* oxygen-evolving complex protein 1
* chloroplast photosynthetic water
oxidation complex
* chloroplast manganese stabilising
protein-II
Responsible for catalyzing the
decomposition of H2O molecules
47 differential spots (NT1xT1): 26 identified proteins
Photosystem II
Marco A. Z. Arruda
Laser ablation Marco A. Z. Arruda
Ablation interaction between the laser and the sample
forming a plasma with surface expansion of the sample and
ejection of the material
Proteins – phenol based extraction(*)
Step Condition Accumulated V
1 0 – 500 V 0.5 kVh
2 500 – 1000 V 0.8 kVh
3 1000 – 10000 V 11.3 kVh
4 10000 V 5.0 kVh
Isoelectric focusing
Reduction (DTT)
Alkylation
MM separation
Marco A. Z. Arruda
Proteins separation: 2D PAGE
(*)
483 ± 13 spots
observed*
73% of correlation
between gels*
*According to ImageMaster 2D Platinum 6.0 software (GeneBio, Geneva , Switzerland)
3 10pI
97.066.0
45.0
30.0
20.1
14.4
kDa
2D-PAGE profile – Sunflower
leaves (Se treated plants)
Marco A. Z. Arruda
Gel 2D PAGE: 18x16 cm2
Portion of the gel:1.5x0.5 cm2
dried under vacuum (75 min at
40 °C), using a filter paper and
plastic
1 mm slab dried gel
Chromatography paper
(Whatman 3MM CHR)
Plastic film
LA-ICP-MS
Optimizing the conditions
Insertion of the gel into the
ablation chamber (New Wave UP-213)
104 spots analyzed
10 Se-containing spots
(9.6%)
Protein identities
Putative alanine aminotransferase
Peroxisomal glycolate oxidase
Glycolate oxidase
Phosphoglycerate kinase
ATP synthase beta subunit
Triose-phosphate isomerase
N-glyceraldehyde-2-phosphotransferase-like
Ribulose-phosphate-epimerase
Triose phosphate isomerase cytosolic isoform-like
Triose-phosphate isomerase
Ribulose bisphosphate carboxylase / RuBisCO
Nucleoside diphosphate kinase
Cu/Zn superoxide dismutase precursor
Thioredoxin peroxidase
Oxygen evolving complex 33 kDa photosystem II protein
Putative peroxidase
Chloroplast heat shock protein
Protein identification
Marco A. Z. Arruda
0 200 400 600 8000
20
40
60
80
100
120
140
160
180
80S
eO
+/1
2C
+ r
atio (
x1000)
Selenium concentration (mg L-1)
0 5 10 15 200.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
32S
16O
+/1
2C
+ R
atio
Sulfur concentration (mg g-1)
(mg/kg)
LA-ICP-MS: quantification
CRM 1575a (Tomate leaves) used for
calibration purposes
CRM 100 (Beech leaves) used for
calibration purposes
Marco A. Z. Arruda
Marco A. Z. Arruda
LA-ICP-MS: results
Spiked
conc.
(mg/kg)
Conc.
digested
(mg/kg)
LA
(mg/kg)
Pellets
CRM 281
800 865±16 886±26
Pellets sunflower
Se treated group
0 703±12 707±36
Pellets sunflower
control group
100 97±9 121±15
Se (n=3)
Certified
value
(mg/g)
Conc.
digested
(mg/g)
LA(mg/g)
Standard additions
method CRM 100
2.69±0.04 --- 2.7±0.1
Pellets
CRM 1575a
0.96* --- 0.99±0.2*
Pellets sunflower Se
treated group
--- 7.6±0.2 6.8±0.6
S (n=3)
*expressed as %
Marco A. Z. Arruda
LA(i)-ICP-MS: identification of Se
(control plants)
50th cultive day
calibration curve (Se): 25 – 850 mg kg-1
50th cultive day
calibration curve (S): 2.7 – 20 mg g-1
Marco A. Z. Arruda
LA(i)-ICP-MS: identification of Se
(treated plants)
45th cultive day after addition of 174 mg Se
Calibration curve (S): 2.7 – 20 mg g-1
45th cultive day after addition of 174 mg Se
Calibration curve (Se): 25 – 1200 mg kg-1
Marco A. Z. Arruda
Unicamp: GEPAM
http://gepam.iqm.unicamp.brAlison
Bruna (1)
Bruna (2)
Cícero
Eraldo
Gustavo
Giovanni
Ivanilce (Téc)
Hugo
Josiane
Katherine
Michele
Mónica
Rodrigo