Marco Aurélio Zezzi ArrudaFull Professor

http://gepam.iqm.unicamp.br

http://www.inctbio.iqm.unicamp.br

e-mail: zezzi@iqm.unicamp.br

Workshop on Interdisciplinary Plant Science

Marco A. Z. Arruda

1) GMOSoybean

2) LA (imaging)Sunflower + Se

Marco A. Z. Arruda

1) GMOSoybean

2) LA (imaging)Sunflower + Se

Marco A. Z. Arruda

Why soybean?

M

hectares

M

acres

PDB information

Expertise

Marco A. Z. Arruda

cp4 EPSPS (5-enolpyruvylshikimate-3-

phosphate synthase) gene from the soil

bacteria Agrobacterium sp. line cp4;

Genetic modification

The transgenic

Our hypothesis:The genetic modification

is acting as stress factor

Marco A. Z. Arruda

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

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-1 Recovery %

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)

Results

Plants

Marco A. Z. Arruda

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 DIGENT2xT2

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

Marco A. Z. Arruda

1) GMOSoybean

2) LA (imaging)Sunflower + Se

Laser ablation Marco A. Z. Arruda

Ablation interaction between the laser and the sampleforming 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)

Se identification – LA-ICP-MS

Marco A. Z. Arruda

Se identification – LA-ICP-MS

Marco A. Z. Arruda

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 %

Silva & Arruda, Metallomics 5(2013)62

LA(i)-ICP-MS

Marco A. Z. Arruda

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

Marco A. Z. Arruda