III Monitoring Visit, DISPAA January 13th 2016
PROJECT LIFE12 ENV/IT/000336
AFTER-Cu
Demonstration of the use of copper compounds for the control
of bacterial diseases of plants important for the EU
ICCOM-CNR Participant personal:
B. Campanella, E. Bramanti, M.
Onor, A. D’Ulivo, E. Pitzalis, M.C.
Mascherpa, S. Biagi, M.Cempini
ICCOM-CNR B2-B7
Consulting Activity for the
demostration Actions
ICCOM-CNR C1-C2-C3
Monitoring Actions Phytormones
dosage
ICCOM-CNR C5
Monitoring Actions Copper
Speciation to evaluate Short and
Long term Envitonmental
Benefits
2
Support to ACTION B1-C1
Study of the effect of the use of copper compounds
2500
Nicotiana shoots (in vitro)
µg/g Cu
µg/g Cu
2000
1500
1000
500
0
0
120
500
8000
7000
6000
5000
4000
3000
2000
1000
0
Nicotiana roots (in vitro)
0
1000
µg/g Cu
µg/g Cu
18
Kiwi (in field)
20
500
1000
CuSO4 (µM)
CuSO4 (µM)
25
120
15
10
Olive (in field)
12
6
5
0
0
0
100
CuSO (µM)
200
0
100
CuSO (µM)
200
Support to ACTION B2-B7and C2-C5
Lyophilized Whole Plants, Roots and Leaves from pilot and in Field
demostration actions
 Quantitation of total copper (Roots and Shoots Distribution)
 Copper speciation
(Adsorbtion, Distribution and Cu Binding
Proteins)
 Effects of Peptides on
Protein Pattern (Cu and other essential
metals)
 Determination of several plant phytohormones related to the
response of vegetable cells to biotic or abiotic stresses
4
Sample Analysis

Determination of Total Cu

Determination of Cu Species (speciation)

Mass Balance of Cu (Trial)

Other Metals (Fe, Mn, Ca, Mg, Zn)
5
Plant pretreatment
TOTAL COPPER
(Cu2+)
Flow
injection
ICP-MS
72Ge
Plant treated with CuSO4
liquid N2
lyophilization
COPPER-BINDING
PROTEINS
(e.g phytochelatin) Zn/Cu
superoxide
dismutase
Size
exclusion
LC
ICP-MS
72Ge
Plant pretreatment
TOTAL COPPER
(Cu2+)
Flow
injection
ICP-MS
72Ge
Plant treated with CuSO4
liquid N2
lyophilization
COPPER-BINDING
PROTEINS
(e.g phytochelatin) Zn/Cu
superoxide
dismutase
Size
exclusion
LC
ICP-MS
72Ge
Intensity (arb. units)
Speciation by SEC – ICP – MS
56Fe
44Ca
24Mg
55Mn
66Zn
63Cu
0
5
10
15
Retention time (min)
20
25
30
30 kDa
75 kDa
6.5 kDa
1.3 kDa
0.9
17 kDa
Kav
exclusion limit
Intensity (arb. units)
1.2
0.6
0.3
0.0
2
3
4
LogMW
5
56Fe
44Ca
24Mg
55Mn
66Zn
63Cu
0
5
10
15
Retention time (min)
20
25
30
6
Examples of metal binding proteins
10
15
20
Olivo
25
1050000
700000
350000
0
330000
220000
110000
0
480000
320000
160000
0
1170000
780000
390000
0
600000
400000
200000
0
10200000
6800000
3400000
0
540
360
180
0
Zn66
Cu63
Fe56
Mn55
Ca44
Mg24
UV 280nm
0
5
10
0
30
15
20
Time (min)
25
30
5
15
20
25
N.10Bethamiana
30
1170000
780000
390000
0
330000
220000
110000
0
261000
174000
87000
0
2190000
1460000
730000
0
720000
480000
240000
0
10200000
6800000
3400000
0
420
280
140
0
Zn66
Cu63
Fe56
Mn55
Ca44
Mg24
UV 280nm
0
5
10
0
15
20
Time (min)
25
30
5
10 Glauca
15
20
N.
25
30
25
1030
Zn66
Cu63
Fe56
Mn55
Ca44
Mg24
UV 280nm
48000
32000
16000
0
111000
74000
37000
0
294000
196000
98000
0
213000
142000
71000
0
162000
108000
54000
0
6300000
4200000
2100000
0
840
560
280
0
5
UV 280nm
J
0
UV 280nm
0
5
10
15
20
Time (min)
NL-GR 6.33
NL-GR 2.4/5
NL-rolC/8
11
Speciation by SEC – ICP – MS
Counts
SOD: a homodimer of
molecular weight 32,500
from bovine red cells
63Cu
profile in
Nicotiana Glauca
300000
Cu-Zn
superoxide
dismutase
225000
150000
75000
0
0
•
5
10
15
Time (min)
The concentrations of copper bound to
proteins can be estimated using the
chromatogram
20
25
30
Basal level of copper and zinc in
non-contaminated plants:
Cu 2 – 4 mg/kg
Zn 40 – 240 mg/kg
Phytohormones determination
Plants secondary metabolites, e.g. phenolic acids and phytohormones, are
involved in a variety of roles in the life span of plants, ranging from
structural ones to protection
Hormones and
phenolic acids
Metabolism
(C & N)
Gene
regulation
Plant growth and
development
Peptide
Biotic and abiotic
stimuli
Copper treatment
13
Phytohormones determination
Solid phase extraction (C18
and HLB cartridges)
Ionic exchange resin
Liquid-liquid
extraction
14
Combination of SPE with liquid-liquid
extraction for the determination of
phytohormones in real samples
Nicotiana langsdorffii
80% methanol-0.1% formic acid
Spike multistandard solutions
50 mM sodium carbonate
Spike multistandard solutions
Magnetic stirring 30 min, 0 °C
HLB
Extraction liquid-liquid
Centrifuged 30 min, 4°C
Extraction liquid-liquid
HLB
15
Extraction in carbonate
(B)
160
SA
160
140
140
140
120
BA
tr = 16.25 min
tr = 16.65 min
120
100
mAU
mAU
120
100
100
80
80
60
60
40
40
15.4
15.6
15.8
16.0
16.2
16.4
16.6
Minutes
16.8
17.0
17.2
17.4
17.6
17.8
18.0
275
250
250
225
225
200
200
3-IAA
tr = 16.03 min
175
150
mV
150
125
125
100
100
3-IAA-Asp
tr = 13.01 min
75
50
NAA
tr = 20.52 min
BAP
120
tr = 14.96 min
100
80
80
60
60
40
40
20
20
13.75 14.00 14.25 14.50 14.75 15.00 15.25 15.50 15.75 16.00 16.25 16.50 16.75 17.00 17.25 17.50 17.75 18.00 18.25 18.50 18.75 19.00 19.25 19.50 19.75 20.00
Minutes
275
175
mV
ABA
tr = 18.60 min
mAU
140
(C)
160
160
mAU
(A)
Representative absorbance at 243 (A) and 273 nm (B) and
fluorescence chromatograms (C), of spiked (red) and
unspiked (black) Nicotiana langsdorffii (NL-rolC/211) extracted
in 50 mM sodium carbonate (λex = 280 nm;
λem = 340
nm).
75
50
25
25
12
13
14
15
16
17
Minutes
18
19
20
21
22
23
16
Molecularly Imprinted Polymers (MIPs)
Synthesis
Acetonitrile
MIP-1
Acetonitrile
Prepolimerization mixture
Methanol
Prepolimerization mixture:
MIP-2
3-IAA and 4-VP -20 °C overnight
MIP-3/NIP-3
17
MIPs/NIP procedure
1
2
Solvent for
rebinding
Standard/sample
MIPs/NIP
MIPs/NIP
Conditioning
3
Sample addition
4
10% methanol
Vortexed 5 min
Centrifuged 10 min
Methanol
MIPs/NIP
MIPs/NIP
Washing step
Eluate injected into HPLC system
18
FL2000/FL3000-340nm
R2_IAA 20ppb in MeOH 5%.dat
FL2000/FL3000-340nm
R8_IAA 20ppb in MeOH5% + MIP1.dat
FL2000/FL3000-340nm
R14_MeOh5%+mip1 rilascio.dat
55
FL2000/FL3000-340nm
R7_MeOH5% + MIP1.dat
50
50
45
45
40
40
IAA 20 ng/g in 5%
methanol
35
mV
FL2000/FL3000-340nm
R3_IAA 20ppb 5% + MIP1RILASCIO.dat
35
30
30
25
25
20
20
MIP-1 washed with
methanol
15
mV
55
15
10
10
IAA treated with
MIP-1
5
5
0
0
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
Minutes
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
10.0
19
Batch rebinding and release experiments
Percentage values relative to the amount of 3-IAA bound to the polymer and the amount released.
The values are expressed as average value ± confidence interval (n=3, LF 95%).
Polymer
3-IAA bound (%)
MIP-1
51 ± 3
51 ± 2
3-IAA released (%)
MIP-3
52 ± 2
95 ± 4
85 ± 2
58 ± 3
88 ± 3 *
60 ± 1 **
70 ± 3
NIP-3
93 ± 3
72 ± 2
MIP-2
* 3 sequential releasing steps
** 10 min ultrasounds treatment during the releasing
test
20
Evaluation of selectivity
Percentage values relative to the amount of SA, ABA, catechin , 3-IAA, IBA, NAA bound to the MIP1 and the amount released. The values are expressed as average value ± confidence interval (n=3,
LF 95%)
Analytes
Catechin
3-IAA
IBA
NAA
SA
ABA
Bound (%)
MIP-1
70 ± 3
50 ± 2
52 ± 4
50 ± 2
50 ± 3
43 ± 2
Release (%)
MIP-1
22 ± 2
83 ± 3
20 ± 3
22 ± 2
29 ± 2
29 ± 3
Bound (%)
NIP-3
74 ± 3
87 ± 2
100 ± 2
93 ± 1
76 ± 3
48 ± 1
Release (%)
NIP-3
100 ± 2
86 ± 1
85 ± 3
71 ± 3
67 ± 2
85 ± 3
21
Application to real samples
Lemon leaves
Lemon leaves infected with Pseudomonas syringae
Nicotiana langsdorffii
1 mL acetonitrile (25 mg plant sample)
1 mL 80% methanol (25 mg plant sample)
1 mL acetonitrile + spike 20 ppb 3-IAA
1 mL 80% methanol + spike 20 ppb 3-IAA
Magnetic stirring 30 min, 0 °C
MIP-1
Centrifuged 30 min, 4°C
NIP-3
22
Nicotiana langsdorffii treated with MIP-1
12
12
3-IAA + spike 20 ppb
tr = 5.84 min
10
8
8
6
6
3-IAA
4
mV
mV
10
4
tr = 5.84 min
2
2
0
0
3.0
3.5
4.0
4.5
5.0
5.5
6.0
Minutes
6.5
7.0
7.5
8.0
8.5
9.0
9.5
Fluorescence chromatogram at 340 nm of plant sample extracts NL-rolC/8 not spiked (black line) and spiked (red
line) with 20 ppb 3-IAA (λex = 280 nm) treated with MIP-1.
23
Nicotiana langsdorffii treated with MIP-1
Fitting parameters of the external and internal calibration curves obtained using MIP-1
Polymer
MIP-1 (internal)
MIP-1 (external)
Concentration range (ppb)
2-20.00
2-20.00
Slope (standard deviation)
6.69·103 ± 100
1.59·104 ± 50
R2
0.99
1.00
Recovery 42 ± 5 %
LOD = 0.65 ± 0.07 ppb (n=3, LF 95%)
LOQ = 2.89 ± 0.08 ppb (n=3, LF 95%)
3-IAA = 6.24 ± 0.04 ppb (n=3, LF 95%)
24
Lemon leaves treated with NIP-3
40
40
35
35
3-IAA + spike
tr = 6.06 min
30
25
mV
mV
25
30
20
20
15
15
3-IAA
tr = 6.06 min
10
10
5
1
5
2
3
4
5
6
7
Minutes
8
9
10
11
12
Fluorescence chromatogram at 340 nm of infected lemon leave extracts not spiked (black curve) and spiked (red
curve) with 20 ppb 3-IAA (λex = 280 nm) treated with NIP-3.
25
Lemon leaves treated with NIP-3
Fitting parameters of the external and internal calibration curves obtained using the purification of lemon
leave infected or not with Pseudomonas syringae with NIP-3
Polymer
Concentration range (ppb)
Slope (standard deviation)
R2
NIP-3 (internal infected)
NIP-3 (internal not infected)
NIP-3 (external)
2.00-40.00
2.00-20.00
2.00-40.00
8.60·103 ± 200
8.80·103 ± 100
1.50·104 ± 90
0.99
0.99
0.99
Recovery 58 ± 5 %
LOD = 0.50 ± 0.08 ppb (n=3, LF 95%)
LOQ = 2.64 ± 0.09 ppb (n=3, LF 95%)
3-IAA = 7.73 ± 0.08 ppb (n=3, LF 95%)
3-IAA = 40.12 ± 0.09 ppb (n=3, LF 95%)
Lemon leaves not infected
Lemon leaves infected
26
Comparison with commercial SPE
3-IAA
tr = 5.75 min
20
18
16
16
14
14
12
12
10
10
mV
mV
18
20
8
8
6
6
4
4
4.6
4.8
5.0
5.2
5.4
5.6
5.8
6.0
6.2
6.4
Minutes
6.6
6.8
7.0
7.2
7.4
7.6
7.8
8.0
8.2
Fluorescence chromatogram at 340 nm of infected lemon leave extracts not spiked (black line) and
spiked (red line) with 20 ppb 3-IAA (λex = 280 nm) treated with HLB cartridges.
27
Carboxylic acids determination by GC-MS
Indole3-acetic
acid
+ Et3O+BF4Benzoic
acid
Salicylic
acid
 1h room
temperature
 extraction
with MTBE
28
28
50 mg of
liophilized
sample
1mL extract or standard
800 μL PBS
250 μL Et3O+ 3.5M
1 ml MTBE
2mL NaOH 150mM
50 μL internal standard
120 μL NaOH/STD_in NaOH 150mM
0.600
0.500
IAA Calibration
y = 0.9793x + 0.001
R² = 0.9984
0.400
0.300
0.200
0.100
0.000
0.000
0.100
0.200
0.300
0.400
0.500
0.600
IAA production by bacteria
Wild type
Tryptophan (1.9 μg/mg
lyophilized bacteria)
IAA-lysine conjugate
R1 9 _ W T N2 1 A1 0 IS O3
350
3 .2 8
300
250
mV
200
2 .6 0
150
100
50
0
0
2.0
4.0
6.0
8.0
M inute s
10.0
12.0
14.0
30
Mass spectrum of
N-ε-(indole-3-acetyl)-l-Lysine
31
Support to ACTION B3 and C2
Conformational study of HrpA peptide linked to
electrode
1.0
1655 1638
128scan_A
1024scan_B
128scan_B
0.6
1546
50
α-Helix
2 β-Turns
42 ap β-sheets
6 ap β-sheets
0.4
Amide I
0.2
1.0
0.0
1800 1750 1700 1650 1600 1550 1500 1450 1400 1350 1300
cm-1
Absorbance, a.u.
Absorbance, a.u.
0.8
HHSBW
Freq. cm-1 cm-1
%
1660
47
1680
17
1627
36
1692
19
-helix
0.8
ap-sheets
0.6
0.4
0.2
-turns
ap-sheets
0.0
1700
1680
1660
cm
1640
1620
1600
-1
mse=3.3*10-7
32
GRAZIE PER L’ATTENZIONE
Emilia
Bramanti
Beatrice
Campanella
Manuela
Cempini
Massimo
Onor
Simona
Biagi
Alessandro
D’Ulivo
Marco Carlo
Marscherpa
Emanuela
Pitzalis
33