Surface-area measurement of polydisperse particles with ...€¦ · Web viewSupplementary Information
11306_2016_999_MOESM1_ESM.docx - Springer …10.1007... · Web viewSupplementary material Alkaloid...
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Supplementary material Alkaloid biosynthesis and metabolic profiling responses to jasmonic acid elicitation in Hamelia patens plants by NMR-based metabolomics Isvett Josefina Flores-Sanchez David Paniagua-Vega Ileana Vera-Reyes Carlos M. Cerda-García-Rojas Ana C. Ramos-Valdivia —————————————————— I. J. Flores-Sanchez D. Paniagua-Vega I. Vera-Reyes A. C. Ramos-Valdivia () Departamento de Biotecnología y Bioingeniería, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Av. IPN 2508, Col. San Pedro Zacatenco, 07360, Mexico, D. F., Mexico.
Transcript of 11306_2016_999_MOESM1_ESM.docx - Springer …10.1007... · Web viewSupplementary material Alkaloid...
Supplementary material
Alkaloid biosynthesis and metabolic profiling responses to jasmonic acid
elicitation in Hamelia patens plants by NMR-based metabolomics
Isvett Josefina Flores-Sanchez David Paniagua-Vega Ileana Vera-Reyes Carlos M. Cerda-
García-Rojas Ana C. Ramos-Valdivia
——————————————————
I. J. Flores-Sanchez D. Paniagua-Vega I. Vera-Reyes A. C. Ramos-Valdivia ()
Departamento de Biotecnología y Bioingeniería, Centro de Investigación y de Estudios Avanzados del Instituto
Politécnico Nacional, Av. IPN 2508, Col. San Pedro Zacatenco, 07360, Mexico, D. F., Mexico.
C. M. Cerda-García-Rojas
Departamento de Química, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Av.
IPN 2508, Col. San Pedro Zacatenco, 07360, Mexico, D. F., Mexico.
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34 4
55
66
7
7
98
10
11
10
12
2
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1
1
14
Fig. S1. Typical 1H-NMR spectra of MeOH/H2O fractions from elicited H. patens plants in the
range of δ1.0–3.2, 4.0–5.7 and 6.2–7.9. Assignments: 1, loganic acid; 2, pteropodine +
isopteropodine; 3, acetic acid; 4, aspartic acid; 5, glutamic acid; 6, glutamine; 7, sucrose; 8, β-
glucose; 9, α-glucose; 10, chlorogenic acid; 11, p-coumaric acid; 12, tryptophan; 13, palmirine;
14, strictosidine.
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A
B
Fig. S2. 1H NMR spectra of the (A) MeOH/H2O and (B) chloroform fractions from elicited
Hamelia patens plants. The extractions were performed by indirect fractionation and the spectra
corresponded to the aromatic region.
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H-β′
H-β
Aspartic acid
H-7′
H-8′
Chlorogenic acid
Fig. S3. COSY spectrum of MeOH/H2O extractions from elicited Hamelia patens plants
confirming the identification of aspartic acid, chlorogenic acid and loganic acid.
5
Loganic acid
H-2′
H-1 ′
Fig. S3. (continued) COSY spectrum of MeOH/H2O extractions from elicited Hamelia patens
plants confirming the identification of aspartic acid, chlorogenic acid and loganic acid.
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Fig. S4. Proposed biosynthesis pathway for pentacyclic indole and oxindole alkaloids in H.
patens. IPP, isopentenyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; GeranylPP,
geranyl pyrophosphate; TDC, tryptophan decarboxylase; DXS, 1-deoxy-D-xylulose-5-phosphate
synthase; SGD, strictosidine β-glucosidase; STR, strictosidine synthase. Dashed arrows,
proposed steps; triplet dot-dash arrows indicate additional steps.
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0
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0.4
0.6
0.8
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3
4
0
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2
3
4
0
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3
4
0.0
0.2
0.4
0.6
0.8
1.0
0 2 4 6 8 10 12 14 16 30 40 91
0.00
0.02
0.04
0.06
0.08
0.10
mol
50 m
g DW
-1
p-Coumaric acid
Tryptophan
Chlorogenic acid
Glucose
Aspartic acid
Sucrose
Acetic acid
*
*
*
**
*
**
*
**
*
*
*
0.0
0.5
1.0
1.5
2.0
2.5
3.0
0 2 4 6 8 10 12 14 16 30 40 91
Loganic acid*
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0 2 4 6 8 10 12 14 16 30 40 91
Time (d)
Strictosidine
** *
Time (d)Time (d)
mol
50 m
g DW
-1m
ol50
mg
DW-1
mol
50 m
g DW
-1
mol
50 m
g DW
-1
Fig. S5. Time course of relative contents of metabolites identified in control (circles) and elicited
(triangles) H. patens plants. TMSP was used as internal standard (0.435 mol). JA application
was made at day 4. Values are expressed as means of 2 ≤ n ≤ 4 with standard deviations. *,
significant difference between metabolite content from control and elicited plants (paired t-test, P
< 0.05).
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0
2
4
6
8
10
12
14
Speciophylline*
*
*
**
**
mg
g D
W-1
0.0
0.4
0.8
1.2
1.6
2.0Rumberine
** *
**
0
5
10
15
20
25
30
**
*
***
mg
g D
W-1
0.0
0.5
1.0
1.5
2.0
2.5
3.0Isopteropodine
**
*
*
*Pteropodine
0.0
0.2
0.4
0.6
0.8
1.0
1.2
0 2 4 6 8 10 12 14 16 30 40 91
Palmirine
0
1
2
3
4
0 2 4 6 8 10 12 14 16 30 40 91
Hameline
**
*
**
*
Time (d)
*
*
*mg
g D
W-1
Time (d)
Fig. S6. Time course of MOA content in control (blue circles) and JA-treated (red triangles)
Hamelia patens plants. Elicitation was applied at day 4. Values are means of 3 ≤ n ≤ 6. *, significant
difference in alkaloid contents between control and JA treatments (pair t-test, P ≤ 0.05).
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0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
0 2 4 6 8 10 12 14 16 30 40 91
Aricine N-oxide
*
*
*
Time (d)
mg
g D
W-1 * *
Aricine
* *
*
**
Tetrahydroalstonine
†
mg
g D
W-1
mg
g D
W-1
Fig. S7. Time course of MIA content in control (blue circles) and JA-treated (red triangles)
Hamelia patens plants. Elicitation was applied at day 4. Values are means of 3 ≤ n ≤ 6. *,
significant difference in MIA contents between control and JA treatments (pair t-test, P ≤ 0.05);
†, not detected.
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In-C
Ox-C
0
5
10
15
20
25
30
0 2 4 6 8 10 12 14 16 30 40 91
mg
g D
W-1
Time (d)
In-C In-JA Ox-C Ox-JA
* ** *
*
*
Δ Δ ΔΔ
Δ
Fig. S8. Time course of MIA and MOA content in control and JA-treated Hamelia patens plants.
Elicitation was applied at day 4. Values are means of 3 ≤ n ≤ 6. *, significant difference in MOA
contents between control and JA treatments; Δ, significant difference in MIA contents between
control and JA treatments (pair t-test, P ≤ 0.05). In-C, MIAs in control treatment; Ox-C, MOAs in
control treatment; In-JA, MIAs in JA treatment; Ox-JA, MOAs in JA treatment.
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CoontrolHpatenst30d #640 RT: 10.31 AV: 1 SB: 23 12.21-12.37, 12.65-12.79 NL: 1.86E7T: + c ESI Full ms [ 50.00-900.00]
50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
385.2
386.2
545.1387.2 529.2388.7 433.9
226.7 804.6304.2 472.9 546.1204.7 646.9
CoontrolHpatenst30d #700 RT: 11.17 AV: 1 SB: 23 12.21-12.37, 12.65-12.79 NL: 3.40E7T: + c ESI Full ms [ 50.00-900.00]
50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
369.2
531.0
370.2325.1
532.0371.2 758.5527.0326.2 533.2 575.0 737.1324.2
Fig. S9. Mass spectra of Hamelia patens alkaloids identified in the MeOH/H2O extractions
obtained by LC-MS. Peak values correspond to [M+H]+.
Rumberine
MW 384
Speciophylline
MW 368
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ElicitedHpatensT30d02 #722 RT: 11.43 AV: 1 SB: 23 12.20-12.37, 12.66-12.78 NL: 4.23E8T: + c ESI Full ms [ 50.00-900.00]
50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
369.2
370.2
385.2
768.8
769.7386.2368.4 736.5160.1
ElicitedHpatensT30d #723 RT: 11.51 AV: 1 SB: 23 12.21-12.38, 12.65-12.78 NL: 3.19E8T: + c ESI Full ms [ 50.00-900.00]
50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900m/z
0
5
10
15
20
25
30
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40
45
50
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60
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70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
369.1
370.2
385.2
386.1 758.8 774.7160.1 736.5
Fig. S9. (continued) Mass spectra of Hamelia patens alkaloids identified in the MeOH/H2O
extractions obtained by LC-MS. Peak values correspond to [M+H]+.
Isopteropodine
MW 368
Pteropodine
MW 368
13
CoontrolHpatenst30d #753 RT: 11.90 AV: 1 SB: 23 12.21-12.37, 12.65-12.79 NL: 1.10E8T: + c ESI Full ms [ 50.00-900.00]
50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900m/z
0
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20
25
30
35
40
45
50
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60
65
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90
95
100
Rel
ativ
e A
bund
ance
399.1
369.2
400.2
401.1 791.1 818.6325.1
CoontrolHpatenst30d #816 RT: 12.79 AV: 1 SB: 23 12.21-12.37, 12.65-12.79 NL: 5.96E7T: + c ESI Full ms [ 50.00-900.00]
50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900m/z
0
5
10
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80
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90
95
100
Rel
ativ
e A
bund
ance
383.1
384.1
174.0750.7 766.7687.3497.1 800.6
Fig. S9. (continued) Mass spectra of Hamelia patens alkaloids identified in the MeOH/H2O
extractions obtained by LC-MS. Peak values correspond to [M+H]+.
Hameline
MW 398
Aricine
MW 382
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ElicitedHpatensT30d02 #842 RT: 13.07 AV: 1 SB: 23 12.20-12.37, 12.66-12.78 NL: 1.32E7T: + c ESI Full ms [ 50.00-900.00]
50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900m/z
0
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45
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Rel
ativ
e A
bund
ance
383.1
796.8
399.1353.1
441.9
800.8
327.3
802.9766.6 803.8
401.2 764.3581.8287.2 810.3492.1 846.7665.7510.4 705.9595.0251.8198.2
ElicitedHpatensT30d #773 RT: 12.18 AV: 1 SB: 23 12.21-12.38, 12.65-12.78 NL: 1.96E7T: + c ESI Full ms [ 50.00-900.00]
310 320 330 340 350 360 370 380 390 400 410 420 430 440m/z
0
5
10
15
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25
30
35
40
45
50
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90
95
100
Rel
ativ
e A
bund
ance
385.1
399.1
369.2
415.2
386.2370.2
387.1326.2 341.5 413.9 433.7 438.8401.1 422.0
Fig. S9. (continued) Mass spectra of Hamelia patens alkaloids identified in the MeOH/H2O
extractions obtained by LC-MS. Peak values correspond to [M+H]+.
Aricine N-oxide
MW 398
Palmirine
MW 398
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ElicitedHpatensT30d #680 RT: 10.91 AV: 1 SB: 23 12.21-12.38, 12.65-12.78 NL: 3.98E8T: + c ESI Full ms [ 50.00-900.00]
50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900m/z
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100
Rel
ativ
e A
bund
ance
531.1
532.1
533.2530.4575.0
Fig. S9. (continued) Mass spectra of Hamelia patens alkaloids identified in the MeOH/H2O
extractions obtained by LC-MS. Peak values correspond to [M+H]+.
Glycosylated oxindole alkaloid
MW 530
