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

Mihail Kantor; Amnon Levi; Judith Thies; Nihat Guner; Camelia Kantor; Stuart Parnham; Arezue Boroujerdi

doi:10.21307/jofnem-2018-030

Figures & Tables

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.

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

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 (H^β,d); 3.8 (H^α,q)	19.0 (C^β); 53 (C^α)
5	Arginine	3.25 (H^δ,t); 1.66, 1.74 (H^γ,m); 1.92 (H^β,m); 3.8 (H^α,t)	43 (C^δ); 27 (C^γ); 30 (C^β); 57 (C^α)
6	Asparagine	2.87, 2.96 (H^β,dd); 4.0 (H^α,q)	37.5 (C^β); 54 (C^α)
7	Aspartate	2.70, 2.82 (H^β,dd); 3.90 (H^α,q)	39.4 (C^β); 55.1 (C^α)
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 (¹CH_2, t); 3.8 (²CH₂, t)	44.1 (¹CH_2, 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 (H^β,m); 2.36 (H^γ,m); 3.76 (H^α,t)	29.8 (C^β); 36.4 (C^γ); 57.4 (C^α)
15	Glutamine	2.16 (H^β,m); 2.5 (H^γ,m); 3.78 (H^α,t)	29.1 (C^β); 34 (C^γ); 57.2 (C^α)
16	Guanidoacetate	3.79 (CH₂, s)	46.3 (CH₂, s)
17	Histamine	3.0 (²CH_2, t);3.3 (¹CH_2, t); 7.10 (²CH, s); 7.86(¹CH,s)	26 (²CH_2, t); 41(¹CH_2, t); 120 (²CH, s); 140 (¹CH,s)
18	Histidine	3.2, 3.2 (H^β,dd); 4.0 (H^α,t); 7.10 (H^δ,s); 7.84 (H^ε,s)	31 (C^β); 58 (C^α); 120 (C^δ); 139 (C^ε)
19	Isoleucine	0.94 (H^δ,t), 1.02 (H^γ,d); 1.2, 1.5 (H^γ,m); 2.0 (H^β,m); 3.7 (H^α,d)	14.0 (C^δ); 17, 6 (C^γ); 39 (C^β); 62 (C^α)
20	Lactate	1.33 (CH₃, d); 4.1 (CH, q)	22.3 (CH₃); 71 (CH)
21	Leucine	0.96, 0.97 (H^δ,d); 1.7 (H^γ,m); 1.7 (H^β,m); 3.7 (H^α,m)	23.8, 24.8 (C^δ); 27 (C^γ); 43 (C^β); 56 (C^α)
22	Lysine	1.4 (H^γ,m); 1.73 (H^δ,m); 1.92 (H^β,m);3.03 (H^ε,t); 3.7 (H^α,t)	24 (C^γ); 29.3 (C^δ); 32.7 (C^β); 42.0 (C^ε); 57 (C^α)
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 (H^β,dq); 3.9 (H^α,q); 7.34 (H^δ,d); 7.39 (H^ζ ,t); 7.43 (H^ε,t)	42 (C^β); 60 (C^α); 132 (C^δ); 131 (H^ζ,t); 132 (H^ε,t);
27	Pyroglutamate	2.04, 2.51 (H^β,m); 2.41 (H^γ,t); 4.18 (H^α,q)	28.2 (C^β);32.5(C^γ); 61.2 (C^α)
28	Raffinose^a	5.44 (^1′CH, d); 5.00 (^1″CH, d)00	95.0 (^1′CH); 101.00 (^1″CH)
29	Sarcosine	2.76 (CH_{3 ,} s); 3.6 (CH_2, s)	35 (CH₃); 53 (CH_2, 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 (H^γ,d); 4.3 (H^β,m); 3.6 (H^α,d)	22.3 (C^γ); 69 (C^β); 63 (C^α)
34	Trimethylamine functional group^b	3.22 (CH₃, s); 3.225 (CH₃, s); 3.23 (CH₃, s)	56.9 (CH₃)
35	Tyrosine	3.07, 3.18 (H^β,dq); 3.9 (H^α,q); 6.91 (H^ε,d); 7.20 (H^δ,d)	42 (C^β); 60 (C^α);120 (C^ε); 134 (C^δ)
36	Valine	1.00, 1.05 (H^γ,d); 2.3 (H^β,m); 3.6 (H^α,d)	19.6, 20.8 (C^γ); 32 (C^β); 63 (C^α)

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 lines^a	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	–
Multiple^b	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

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

Figures & Tables

Fig. 1

Fig. 2

Supplemental Fig. 1

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

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

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

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

Paradigm

My account