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NMR Analysis Reveals a Wealth of Metabolites in Root-Knot Nematode Resistant Roots of Citrullus amarus Watermelon Plants

Mihail Kantor
Mihail Kantor
, 
Amnon Levi
Amnon Levi
, 
Judith Thies
Judith Thies
, 
Nihat Guner
Nihat Guner
, 
Camelia Kantor
Camelia Kantor
, 
Stuart Parnham
Stuart Parnham
 oraz 
Arezue Boroujerdi
Arezue Boroujerdi
   | 17 paź 2018
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Data publikacji: 17 paź 2018
Zakres stron: 303 - 316
DOI: https://doi.org/10.21307/jofnem-2018-030
Słowa kluczowe
© 2018 Mihail Kantor et al., published by Sciendo
This work is licensed under the Creative Commons Attribution 4.0 International License.

Fig. 1

Pair-wise comparison between Citrullus amarus PIs and Citrullus lanatus Charleston Gray (CG), respectively Crimson Sweet (CSW). PC1 vs. PC2 score plots are shown for each pairwise comparison with a total explained variance of >70% for each. The ovals represent 95% confidence intervals, and the Mahalanobis distances (M D) are listed for each.
Pair-wise comparison between Citrullus amarus PIs and Citrullus lanatus Charleston Gray (CG), respectively Crimson Sweet (CSW). PC1 vs. PC2 score plots are shown for each pairwise comparison with a total explained variance of >70% for each. The ovals represent 95% confidence intervals, and the Mahalanobis distances (M D) are listed for each.

Fig. 2

Pathway analysis using significantly different metabolites involved 9 metabolic pathways (shown inside rounded boxes). Yellow highlighted metabolites correspond to those listed in Table 3.
Pathway analysis using significantly different metabolites involved 9 metabolic pathways (shown inside rounded boxes). Yellow highlighted metabolites correspond to those listed in Table 3.

Supplemental Fig. 1

Unknown peak assignments using one dimensional 1H and two dimensional 1H–13C HSQC NMR. The 1H spectrum is shown in blue and is at the top of the 1H–13C HSQC shown with red contour points. Each contour point of the HSQC represents a hydrogen directly bounded to a carbon. This specific HSQC experiment shows red contour points for CH or CH3 correlations and green contour points for CH2 correlations.
Unknown peak assignments using one dimensional 1H and two dimensional 1H–13C HSQC NMR. The 1H spectrum is shown in blue and is at the top of the 1H–13C HSQC shown with red contour points. Each contour point of the HSQC represents a hydrogen directly bounded to a carbon. This specific HSQC experiment shows red contour points for CH or CH3 correlations and green contour points for CH2 correlations.

List of metabolites identified in Citrullus amarus or Citrullus lanatus by using 1D 1H and 2D 1H–13C-NMR spectroscopy. All chemical shifts for H–C pairs in each metabolite are given with bold chemical shifts corresponding to the peaks labeled in the 1D 1H noesy and 2D 1H–13C HSQC spectra shown in figure x. Un-bold chemical shift estimations are given for H–C pairs that were not identified in the spectra due to overlap with other peaks or noise.

No. Metabolite 1H chemical shifts (ppm) (functional group or specific H, multiplicity of peak) 13C chemical shifts (ppm) (functional group or specific C)
1 4-Aminobutyrate 1.92 (2CH2); 2.30 (1CH2); 3.02 (3CH2) 26.6 (2CH2); 37.2 ( 1CH2);42.0 (3CH2)
2 2-Oxoglutarate 2.50 (1CH2, t); 3.02 (2CH2, t) 33 (1CH2); 39 (2CH2)
3 Acetate 1.92 (CH3, s) 26 (CH3)
4 Alanine 1.48 (Hβ,d); 3.8 (Hα,q) 19.0 (Cβ); 53 (Cα)
5 Arginine 3.25 (Hδ,t); 1.66, 1.74 (Hγ,m); 1.92 (Hβ,m); 3.8 (Hα,t) 43 (Cδ); 27 (Cγ); 30 (Cβ); 57 (Cα)
6 Asparagine 2.87, 2.96 (Hβ,dd); 4.0 (Hα,q) 37.5 (Cβ); 54 (Cα)
7 Aspartate 2.70, 2.82 (Hβ,dd); 3.90 (Hα,q) 39.4 (Cβ); 55.1 (Cα)
8 Betaine 3.27 (CH3, s); 3.91 (CH2, s) 56 (CH3); 69 (CH2)
9 Choline 3.21 (CH3, s); 3.52 (1CH2, m); 4.07 (2CH2, m) 56.8 (CH3); 70.3 (1CH2); 58.5 (2CH2)
10 Ethanolamine 3.15 (1CH2, t); 3.8 (2CH2, t) 44.1 (1CH2, t); 60 (2CH2, t);
11 Formate 8.46 (CH, s) 174 (CH)
12 Fructose 3.57, 3.72 (1CH2, dd); 3.70, 4.03 (6CH2, dd); 3.80 (3CH, d); 3.8,4.00 (5CH, d); 3.90, 4.12 (4CH, t) 65.3 (1CH2); 66.2 (6CH2); 70.5 (3CH); 70 (5CH); 72.5 (4CH)
13 Glucose 3.25 (2βCH, dd), 3.41 (4CH, dd); 3.47 (5βCH, m); 3.50 (3βCH, t); 3.54 (2αCH, dd); 3.72 (3αCH, t); 3.73 (6βCH2, dd), 3.84 (5αCH, m); 3.90 (6αCH2, dd); 4.65 (1αCH, d); 5.24 (1βCH,d) 77.0 (2βCH); 72.5 (4CH); 78.8 (5βCH); 78.7 (3βCH); 74.3 (2αCH); 75.7 (3αCH); 63.7 (6CH); 74.4 (5αCH); 98.8 (1αCH); 94.9 (1βCH)
14 Glutamate 2.07, 2.14 (Hβ,m); 2.36 (Hγ,m); 3.76 (Hα,t) 29.8 (Cβ); 36.4 (Cγ); 57.4 (Cα)
15 Glutamine 2.16 (Hβ,m); 2.5 (Hγ,m); 3.78 (Hα,t) 29.1 (Cβ); 34 (Cγ); 57.2 (Cα)
16 Guanidoacetate 3.79 (CH2, s) 46.3 (CH2, s)
17 Histamine 3.0 (2CH2, t);3.3 (1CH2, t); 7.10 (2CH, s); 7.86(1CH,s) 26 (2CH2, t); 41(1CH2, t); 120 (2CH, s); 140 (1CH,s)
18 Histidine 3.2, 3.2 (Hβ,dd); 4.0 (Hα,t); 7.10 (Hδ,s); 7.84 (Hε,s) 31 (Cβ); 58 (Cα); 120 (Cδ); 139 (Cε)
19 Isoleucine 0.94 (Hδ,t), 1.02 (Hγ,d); 1.2, 1.5 (Hγ,m); 2.0 (Hβ,m); 3.7 (Hα,d) 14.0 (Cδ); 17, 6 (Cγ); 39 (Cβ); 62 (Cα)
20 Lactate 1.33 (CH3, d); 4.1 (CH, q) 22.3 (CH3); 71 (CH)
21 Leucine 0.96, 0.97 (Hδ,d); 1.7 (Hγ,m); 1.7 (Hβ,m); 3.7 (Hα,m) 23.8, 24.8 (Cδ); 27 (Cγ); 43 (Cβ); 56 (Cα)
22 Lysine 1.4 (Hγ,m); 1.73 (Hδ,m); 1.92 (Hβ,m);3.03 (Hε,t); 3.7 (Hα,t) 24 (Cγ); 29.3 (Cδ); 32.7 (Cβ); 42.0 (Cε); 57 (Cα)
23 Malate 2.37, 2.67 (CH2, dd); 4.30 (CH, dd) 45.5 (CH2); 73.3 (CH)
24 Malonate 3.15 (CH2, s) 50 (CH2)
Methanol (contaminant) 3.36 (CH3, s) 51 (CH3, s)
25 Oxalacetate 2.41 (CH2, s) 28.4 (CH2)
26 Phenylalanine 3.12, 3.29 (Hβ,dq); 3.9 (Hα,q); 7.34 (Hδ,d); 7.39 (Hζ ,t); 7.43 (Hε,t) 42 (Cβ); 60 (Cα); 132 (Cδ); 131 (Hζ,t); 132 (Hε,t);
27 Pyroglutamate 2.04, 2.51 (Hβ,m); 2.41 (Hγ,t); 4.18 (Hα,q) 28.2 (Cβ);32.5(Cγ); 61.2 (Cα)
28 Raffinosea 5.44 (1′CH, d); 5.00 (1″CH, d)00 95.0 (1′CH); 101.00 (1″CH)
29 Sarcosine 2.76 (CH3 , s); 3.6 (CH2, s) 35 (CH3); 53 (CH2, s)
30 Succinate 2.40 (CH2, s) 37.1 (CH2)
31 Sucrose 3.49 (3CH, t); 3.6 (2CH, dd); 3.69 (1′CH2, s); 3.77 (4CH, t); 3.85 (5CH, m); 3.83 (6,6′CH2, m); 3.90 (5′CH, m); 4.06 (4′CH, t); 4.23 (3′CH, d); 5.42 (1CH, d) 72.2 (3CH); 74 (2CH); 64.3 (1′CH2); 75.6 (4CH); 75.3 (5CH); 65.4 (6,6′CH2); 84.2 (5′CH); 76.9 (4′CH); 79.4 (3′CH); 94.9 (1CH)
32 Tartrate 4.35 (CH, s) 77 (CH)
33 Threonine 1.33 (Hγ,d); 4.3 (Hβ,m); 3.6 (Hα,d) 22.3 (Cγ); 69 (Cβ); 63 (Cα)
34 Trimethylamine functional groupb 3.22 (CH3, s); 3.225 (CH3, s); 3.23 (CH3, s) 56.9 (CH3)
35 Tyrosine 3.07, 3.18 (Hβ,dq); 3.9 (Hα,q); 6.91 (Hε,d); 7.20 (Hδ,d) 42 (Cβ); 60 (Cα);120 (Cε); 134 (Cδ)
36 Valine 1.00, 1.05 (Hγ,d); 2.3 (Hβ,m); 3.6 (Hα,d) 19.6, 20.8 (Cγ); 32 (Cβ); 63 (Cα)

Germplasm lines of Citrullus amarus (CA) and watermelon cultivars (Citrullus lanatus; CL) used in this study.

Watermelon germplasm line/cultivar Parental PI of germplasm line Citrullus spp.
W2001 PI 482303 CA
W1832 PI 189225 CA
W1849 PI 482324 CA
W1254 PI 244017 CA
W1446 PI 244018 CA
W1813 PI 482319 CA
W1482 PI 482259 CA
Charleston Gray CL
Crimson Sweet CL

Significantly different metabolites in each of the Citrullus amarus PIs when compared with Citrullus lanatus Charleston Gray (CG), respectively Crimson Sweet (CSW).

1832 1254 1849 1813 1446 1482 2001
Metabolites with higher concentrations in resistant linesa Crimson Sweet (CSW), Fold Change or Charleston Grey (CG), Fold Change
2-oxoglutarate + 4-aminobutyrate – – – – – CSW 6.27 –
4-aminobutyrate – – CSW 5.42 – – – CSW 6.23 CG 1.66
Alanine CSW 3.14 – CSW 4.53 CG 4.56 – CSW 2.11 CSW 4.02 CG 1.53 CSW 4.43 CG 1.78
Arginine CSW 3.4 CG 2.50 – – – – – –
Asparagine CSW 47.72 CG 21.47 – – – – – –
Betaine CSW 5.87 – – – – – –
Choline CSW 2.86 CG 1.76 – CSW 2.40 CG 1.50 – CSW 1.63 – CSW 2.32 CG 1.41
Ethanolamine CSW 5.77 CG 1.86 – CSW 4.11 – – – CSW 3.77
Formate – – – – CSW 2.51 CG 3.07 – –
Fructose – CSW 2.83 CG 1.97 CSW 2.38 CG 1.85 – CSW 2.36 CSW 3.83 CG 2.33 CSW 2.79 CG 1.71
Glucose CSW 4.28 CSW 2.19 CSW 2.10 – CSW 1.87 CSW 2.82 CG 1.37 CSW 3.81 CG 1.26
Glutamate CG 2.29 – CSW 15.60 – – – CSW 26.02
Glutamine CSW 5.17 CG 3.09 – CSW 6.77 CG 5.40 – CSW 3.81 CG 2.32 CSW 3.81 CG 2.22 CSW 3.14 CG 2.18
Isoleucine CG 2.59 – CSW 7.18 – – – CSW 8.73 CG 2.55
Lysine – – – – – – CG 14.09
Malate – CSW 20.6 CG 1.75 CSW 2.47 CSW 3.58 CSW 21.94 CG 1.97 CSW 9.79 CG 1.61 –
Malonate + ethanolamine – – – – – CSW 2.29 –
Multipleb CSW 2.27 – CSW 2.16 – – – CSW 2.37
Raffinose – – – – – – CSW 2.60 CG 2.22
Succinate – CSW 1.94 – – CSW 3.23 CG 2.04 – –
Succinate/Oxalacetate – – CSW 1.92 – – CSW 1.89 –
Sucrose – – – – – CG 2.96 CSW 4.04 CG 3.67
Sucrose/Choline – – – – – CSW 2.95 –
Tartaric acid – – – – – CSW 2.20 –
Trimethylamine functional group CG 2.72 – CG 1.30 CSW 1.86 CG 1.71
UNK at 1.22 – – CSW 1.88 – – – –
UNK at 6.41 CG 5.07 – CSW 7.34 CG 7.49 CSW 5.94 CG 6.12 – CSW 5.05 CG 4.92 CSW 4.94 CG 5.05
UNK at 7.64 CSW 4.28 CG 5.40 – CSW 7.08 CG 8.24 CG 4.82 – CSW 4.20 CG 5.00 CSW 4.32 CG 5.52
UNK at 7.68 CG 3.61 – CSW 2.08 CG 3.49 CSW 4.89 CG 4.12 CSW 1.91 CG 2.21 CG 3.02
Valine CSW 9.94 CG 3.58 – CSW 9.47 – – CSW 5.33 CG 3.07 CSW 10.22 CG 2.59

Consensus of metabolites that have significantly higher concentrations in the Citrullus amarus PIs compared with the Citrullus lanatus cultivars.

CA PI versus cultivars
4-Aminobutyrate
Alanine
Arginine
Asparagine
Ethanolamine
Formate
Fructose
Glutamate
Glutamine
Isoleucine
Succinate/oxalacetate
Unk 6.14
Unk 7.64
Unk 7.68
Valine

Statistical analysis and significance levels of metabolic change in Citrullus amarus PIs as compared with Citrullus lanatus Crimson Sweet (CSW) and Charleston Gray (CG).

Statistical Parameter 1254 vs. CSW 1446 vs. CSW 1482 vs. CSW 1813 vs. CSW 1832 vs. CSW 1849 vs. CSW 2001 vs. CSW
D M 4.43 4.17 4.57 3.76 7.35 14.94 2.43
F-true 26.48 23.49 28.24 19.09 72.91 301.50 7.95
F-critical 4.96 4.96 4.96 4.96 4.96 4.96 4.96
Significance status Yes Yes Yes Yes Yes Yes Yes
Statistical Parameter 1254 vs. CG 1446 vs. CG 1482 vs. CG 1813 vs. CG 1832 vs. CG 1849 vs. CG 2001 vs. CG
D M 6.19 3.41 4.12 5.51 7.14 4.90 5.27
F-true 51.74 15.73 22.93 41.02 68.76 32.37 37.48
F-critical 4.96 4.96 4.96 4.96 4.96 4.96 4.96
Significance status Yes Yes Yes Yes Yes Yes Yes
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