Workshop on Interdisciplinary Plant Science

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1 Workshop on Interdisciplinary Plant Science Marco Aurélio Zezzi Arruda Full Professor

2 1) GMO Soybean 2) LA (imaging) Sunflower + Se

3 1) GMO Soybean 2) LA (imaging) Sunflower + Se

4 Why soybean? M acres M hectares PDB information Expertise

5 The transgenic Genetic modification cp4 EPSPS (5-enolpyruvylshikimate-3- phosphate synthase) gene from the soil bacteria Agrobacterium sp. line cp4;

6 Our hypothesis: The genetic modification is acting as stress factor

7 Results Seeds Transgenic soybean: Roundup Ready Varieties: MSOY 7575 RR (transgenic T) MSOY 7501 (non transgenic NT)

8 Proteomic profile: 4-7 ph range 192 identified proteins Identification of the CP4 EPSPS involved in the genetic modification

9 Proteomic profile: 2-D DIGE Actin (fragment) -Cellular motility -Bind ATP and others proteins Glycine-rich RNA-binding protein -Involved in the cellular response to the environment and development conditions Cytosolic glutamine synthetase -Expressed in roots -Binding to ATP -Fixation of N -Biosynthesis of glutamine Glycinin subunit G1 -Nutritional, physical-chemical, physiological properties of seed

10 Enzymatic activity Soybean seeds APX 1 CAT 2 GR 3 SOD 4 T 18.84± ± ± ±1.81 NT 13.08± ± ± ±3.11 T/NT mm ascorbate.min -1 mg protein -1 ; 2 nm H 2 O 2.min -1 mg protein -1 ; 3 μm GSSH.min -1 mg protein -1 ; 4 units of SOD.mg protein -1 Lipid peroxidation MDA (nm/g): T 2.25±0.04 NT 1.56±0.06 Seeds present a considerable stress!!

11 Total determination 200 mg seeds Varieties MSOY 7501 and MSOY7575RR Dry to constant mass 6.3 ml HNO ml H 2 O 2 Microwave assisted decomposition (35 min)

12 Total determination Mataveli et al., Metallomics, 2(2010)800 a LOD b LOQ Soybean seeds, µg g -1 T NT Al 0.08±0.04 <0.24 a As < a < a Ba 0.104±0.004 <0.002 b Bi <0.001 a <0.001 a Ca 935±37 859±36 Cd < b < b Co 0.045± ±0.001 Cr <0.15 a <0.15 a Cu 10.3± ±0.8 Fe 81.2± ±2.4 Hg < a < a Li <0.006 a <0.006 a Mg 3684± ±507 Mn 28.1± ±2.3 Mo 0.31± ±0.06 Ni 0.49± ±0.08 P 6645± ±718 Pb 0.006± ±0.003 S 3006± ±242 Sb 0.001± ±0.001 Se 0.11±0.04 <0.02 b Sn <0.031 a <0.031 a Sr 21.2± ±3.3 Te < a < a Zn 41.3± ±1.7

13 Bioaccessibility test* 0.3 g seeds + 5 ml gastric solution (pepsine + NaCl) Incubation 37 4h 10 ml de intestinal solution (pancreatin + amylase + biliary salts) Incubation 37 4h Centrifugation (ICP-SF-MS) *Mounicou, S., et al., J. Anal. At. Spectrom., 17(2002)880

14 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) Concentration, µg g -1 Recovery % Bioaccessible fraction Residues T NT T NT T NT Ca 824± ±72 160±17 193±8 105± ±12.7 Cu 3.52± ± ± ± ±8.1 85±8.6 Fe 29.1± ± ± ± ± ±6.8 Mg 1690± ±78 492±53 511±20 91± ±7.6 Mn 12.2± ± ± ± ±9.0 73±10.0 S 1322± ±76 958± ±68 73± ±9.6 Zn 22.1± ± ± ±0.8 92± ±7.6

15 Results Plants

16 Soybean cultivation Plants cultivated in BOD chamber 27±0.1 o C Photoperiod: 12h Substrate for plants (BasaPlanta+Vermflock agro) Water supply in alternate days Germination: ca. 3 days Leaves were harvest at 10 th (T1) and 15 th (T2) days of cultivation (but not from the same plant)

17 Enzymatic activity Leaves APX CAT GR SOD T1 546± ± ±0, ±0.036 T2 429± ± ±0, ±0.027 NT1 431± ± ±0, ±0.020 NT2 414± ± ± ±0.028 T1/NT (21%) 1.16 (14%) 3.35 (70%) 1.45 (31%) T2/NT (3.5%) 1.35 (26%) 4.00 (75%) 1.08 (7.3%) T1 10 th day T2 15 th day 1 mm ascorbate.min -1 mg protein -1 ; 2 nm H 2 O 2.min -1 mg protein -1 ; 3 μm GSSH.min -1 mg protein -1 ; 4 units of SOD.mg protein -1

18 MDA (µm/g fresh material) Hydrogen peroxide (mmol/l) Hydrogen peroxide 0, ± ,02 0,015 0, ± ± ± ,005 0 NT 1 NT 2 T1 T2 Lipid peroxidation 0,8 0,7 0,6 0,5 0,4 0,3 0,2 0, ± ± ± ±0.001 NT 1 NT 2 T1 T2 Leaves present considerable stress!!

19 Proteomic profile: 2-D DIGE NT1 T1 NT1 T1 NT1xT1 NT1 T1 NT1 T1 NT1 T1 NT1 T1 47 differential spots: 19 more abundant (blue) 28 less abundant (red)

20 Proteomic profile: 2-D DIGE NT2 T2 NT2xT2 NT2 T2 NT2 T2 NT2 T2 NT2 T2 NT2 T2 28 differential spots: 25 more abundant (blue) 3 less abundant (red)

21 Differential protein species 47 differential spots (NT1xT1): 26 identified proteins * oxygen-evolving complex protein 1 * chloroplast photosynthetic water oxidation complex * chloroplast manganese stabilising protein-ii Photosystem II Responsible for catalyzing the decomposition of H 2 O molecules

22 1) GMO Soybean 2) LA (imaging) Sunflower + Se

23 Laser ablation Ablation interaction between the laser and the sample forming a plasma with surface expansion of the sample and ejection of the material

24 Proteins separation: 2D PAGE Proteins phenol based extraction (*) Isoelectric focusing Step Condition Accumulated V V 0.5 kvh V 0.8 kvh V 11.3 kvh V 5.0 kvh Reduction (DTT) Alkylation MM separation (*)

25 2D-PAGE profile Sunflower leaves (Se treated plants) kda 3 pi ± 13 spots observed* 73% of correlation between gels* 14.4 *According to ImageMaster 2D Platinum 6.0 software (GeneBio, Geneva, Switzerland)

26 LA-ICP-MS Optimizing the conditions Insertion of the gel into the ablation chamber (New Wave UP-213) Gel 2D PAGE: 18x16 cm 2 Portion of the gel:1.5x0.5 cm 2 dried under vacuum (75 min at 40 C), using a filter paper and plastic Plastic film 1 mm slab dried gel Chromatography paper (Whatman 3MM CHR)

27 Se identification LA-ICP-MS

28 Se identification LA-ICP-MS

29 Protein identification 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

30 LA-ICP-MS: quantification ratio (x1000) 80 SeO + / 12 C Selenium concentration (mg (mg/kg) L -1 ) CRM 1575a (Tomate leaves) used for calibration purposes Ratio 32 S 16 O + / 12 C Sulfur concentration (mg g -1 ) CRM 100 (Beech leaves) used for calibration purposes

31 LA-ICP-MS: results Se (n=3) Pellets CRM 281 Pellets sunflower Se treated group Pellets sunflower control group Spiked conc. (mg/kg) Conc. digested (mg/kg) LA (mg/kg) ±16 886± ±12 707± ±9 121±15 S (n=3) Standard additions method CRM 100 Pellets CRM 1575a Pellets sunflower Se treated group Certified value (mg/g) Conc. digested (mg/g) LA(mg/g) 2.69± ± * ±0.2* ± ±0.6 *expressed as %

32 LA(i)-ICP-MS Silva & Arruda, Metallomics 5(2013)62

33 LA(i)-ICP-MS: identification of Se (control plants) 50 th cultive day calibration curve (Se): mg kg th cultive day calibration curve (S): mg g -1

34 LA(i)-ICP-MS: identification of Se (treated plants) 45 th cultive day after addition of 174 mg Se Calibration curve (Se): mg kg th cultive day after addition of 174 mg Se Calibration curve (S): mg g -1

35 Alison Bruna (1) Bruna (2) Cícero Eraldo Gustavo Giovanni Unicamp: GEPAM Ivanilce (Téc) Hugo Josiane Katherine Michele Mónica Rodrigo

36

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