Table 1.

Identified metabolites of L. japonicus nodules harvested from plants grown under normal photoperiod (N. Ph.), or subjected to 24h and 72h continuous darkness.

	m\z	RI	PC1 (49%) loadings	PC2 (27%) loadings	N. Ph. average response	24h darkness average response	72h darkness average response	24h/N. Ph. response ratio	72h/N. Ph. response ratio	P-value
Sugars
Sucrose	361	2385	–0.5010	0.3580	8.679	7.866	3.276	0.91 a	0.38 b	<0.001
Raffinose	87	3056	–0.0197	–0.0103	0.268	0.060	0.026	0.22	0.10	0.010
Maltose	361	2495	0.0005	–0.0023	0.059	0.051	0.068	0.86	1.14	0.833
Trehalose	361	2505	0.0144	–0.0178	0.329	0.413	0.492	1.25	1.49	0.310
d-Melezitose	361	3153	–0.0380	–0.0010	0.558	0.231	0.069	0.41 a	0.12 b	<0.001
Melibiose	361	2512	–0.0046	0.0043	0.216	0.146	0.155	0.67	0.72	0.208
Cellobiose	361	2252	–0.0002	–0.0006	0.020	0.016	0.015	0.79	0.77	0.870
Maltotriose	361	3034	–0.0031	0.0008	0.039	0.015	0.002	0.40	0.06	0.012
d-Lyxose	217	1389	–0.0012	0.0001	0.024	0.019	0.011	0.80	0.48	0.393
d-Glucose	319	1614	–0.0187	0.0033	0.237	0.088	0.000	0.37 a	0.00 b	0.047
Amino acids
l-Cysteine	220	1250	0.0047	–0.0030	0.000	0.042	0.047	Dark sp.	Dark sp.	0.529
l-Ornithine	142	1310	0.0150	–0.0091	0.000	0.000	0.185	ND	Dark sp.	–
l-Homoserine	218	1145	0.0103	0.0018	0.017	0.062	0.129	3.70	7.74	0.028
l-Proline	142	983	–0.0048	0.0025	0.106	0.074	0.042	0.69	0.40	0.603
l-Asparagine	231	1376	0.4410	0.1160	11.782	14.955	17.094	1.27 a	1.45 b	<0.001
β-Alanine	248	1116	0.0057	–0.0005	0.052	0.060	0.119	1.16 a	2.30 b	0.009
l-Glutamic acid	246	1319	0.1840	0.8760	2.066	5.274	4.310	1.28	1.15	0.211
β-Cyano-l-alanine	141	1064	0.0022	0.0020	0.190	0.201	0.187	1.06	0.98	0.959
l-Alanine	116	798	–0.0252	0.0160	0.400	0.397	0.119	0.99 a	0.30 b	0.004
l-Serine	204	1055	0.0262	–0.0019	0.330	0.430	0.673	1.31 a	2.04 b	0.018
l-Aspartic acid	232	1216	0.3470	–0.0640	0.873	2.162	4.931	2.48 a	5.65 b	<0.001
l-Lysine	317	1618	0.0722	–0.0294	0.212	0.334	1.066	1.57 a	5.02 b	<0.001
l-Tyrosine	218	1635	0.0674	0.0217	0.000	0.682	0.778	Dark sp.	Dark sp.	0.059
l-Methionine	176	1208	0.0066	–0.0036	0.000	0.000	0.070	ND.	Dark sp.	–
l-Histidine	154	1618	0.1240	–0.0340	0.065	0.186	1.450	2.88 a	22.45 b	<0.001
l-Valine	144	903	0.0888	–0.0378	0.106	0.280	1.185	2.65 a	11.21 b	<0.001
l-Threonine	218	1083	0.0211	–0.0002	0.166	0.343	0.434	2.07	2.62	<0.001
l-Tryptophan	202	1918	0.0878	–0.0262	0.046	0.350	1.120	7.64 a	24.41 b	<0.001
dl-Isoleucine	158	982	0.0768	0.0058	0.034	0.297	0.875	8.70 a	25.58 b	<0.001
l-Glutamine	156	1470	–0.0184	–0.0036	0.411	0.129	0.130	0.31	0.32	<0.001
Glycine	174	993	0.0103	0.0021	0.027	0.097	0.146	3.55	5.35	0.028
Organic acids
Dehydroascorbic acid	173	1553	–0.0025	–0.0019	0.036	0.000	0.000	0.00	0.00	–
Shikimic acid	204	1517	–0.0019	0.0003	0.032	0.000	0.000	0.00	0.00	–
6-Hydroxycaproic acid	147	1010	0.0017	–0.0011	0.018	0.031	0.033	1.68	1.81	0.626
l-Ascorbic acid	332	1659	–0.0107	–0.0086	0.173	0.041	0.000	0.24	0.00	0.039
l-Lactic acid	147	765	–0.0097	–0.0093	0.301	0.306	0.206	1.02	0.69	0.519
Succinic acid	148	999	–0.0241	0.0232	0.541	0.677	0.250	1.25 a	0.46 b	0.006
Pyruvic acid	174	756	–0.0010	–0.0000	0.020	0.012	0.005	0.61 a	0.27 b	<0.001
Glyceric acid	189	1024	0.0025	0.0005	0.036	0.038	0.067	1.05	1.87	0.165
Pipecolic acid	156	1051	0.0139	0.0068	0.144	0.171	0.275	1.18	1.90	0.263
Mucic acid	333	1763	0.0003	0.0009	0.317	0.348	0.317	1.10	1.00	0.387
Citric acid	347	1529	–0.1140	0.0781	8.026	7.309	6.357	0.91	0.79	0.069
Isocitric acid	273	1531	0.0023	0.0029	0.000	0.079	0.056	Dark sp.	Dark sp.	0.537
Citramalic acid	147	1169	0.1520	0.1640	5.783	8.406	7.776	1.45	1.34	0.005
Maleic acid	147	989	–0.0018	–0.0019	0.048	0.055	0.025	1.15	0.52	0.502
Fumaric acid	245	1033	–0.0020	0.0002	0.159	0.150	0.119	0.94	0.75	0.187
Glucaric acid	333	1732	–0.0017	0.0010	0.480	0.439	0.434	0.92	0.90	0.843
Gluconic acid	333	1698	–0.0079	0.0038	0.459	0.213	0.255	0.46	0.56	0.034
2-Hydroxycinnamic acid	293	1397	0.0112	–0.0026	0.668	0.884	0.777	1.32	1.16	0.298
4-Hydroxycinnamic acid	293	1624	–0.0011	–0.0023	0.038	0.042	0.015	1.09	0.40	0.464
Citraconic acid	147	1040	–0.0000	0.0001	0.002	0.004	0.001	2.32	0.77	0.437
Glycolic acid	147	775	–0.0008	0.0002	0.023	0.012	0.008	0.53	0.35	0.021
Malonic acid	147	890	–0.0094	0.0005	0.736	0.732	0.671	1.00	0.91	0.926
d-Malic acid	147	1187	–0.3360	0.1160	5.901	5.240	1.798	0.89 a	0.30 b	<0.001
Oxalic acid	147	824	–0.0130	0.0027	0.325	0.272	0.177	0.84 a	0.54 b	0.013
Polyols
Galactinol	204	2708	–0.0070	–0.0037	0.092	0.031	0.010	0.33	0.11	0.101
Palatinitol	361	2555	0.0011	0.0009	0.034	0.036	0.053	1.06	1.56	0.417
d-Mannitol	319	1648	0.0471	0.1800	0.477	1.643	1.161	3.45	2.43	0.020
Pinitol	260	1550	–0.4390	–0.0313	22.215	22.108	18.608	1.00	0.84	0.223
allo-Inositol	318	1745	–0.0008	–0.0010	0.216	0.202	0.201	0.94	0.93	0.984
Phosphates
O-Phospho-l-threonine	370	1534	–0.0026	–0.0008	0.037	0.000	0.000	0.00	0.00	–
Phosphoric acid	299	967	–0.0072	0.0056	3.422	2.796	3.168	0.82	0.93	0.245
Phosphoenolpyruvic acid	369	1302	0.0014	–0.0000	0.008	0.012	0.028	1.57 a	3.68 b	0.007
O-Phosphocolamine	299	1485	0.0161	–0.0036	0.010	0.089	0.203	8.79 a	19.97 b	<0.001
Pyrophosphate	451	1377	–0.0013	0.0001	0.021	0.009	0.003	0.41	0.16	<0.001
Adenosine-5-monophosphate	315	2751	–0.0188	–0.0008	0.366	0.161	0.111	0.44 a	0.30 b	<0.001
3-Phosphoglycerate	357	1519	0.0015	–0.0044	0.116	0.018	0.134	0.16	1.16	0.291
3-Phosphoglyceric acid	387	1519	0.0028	0.0034	0.086	0.096	0.132	1.12	1.53	0.232
d-Glucose-6-phosphate	387	2052	–0.0221	–0.0051	0.348	0.144	0.098	0.41	0.28	0.019
Uridine 5’-monophosphate	315	2563	–0.0029	–0.0003	0.044	0.074	0.030	1.65	0.69	0.181
Glycerol 1-phosphate	357	1471	–0.0011	–0.0011	0.155	0.151	0.126	0.97	0.82	0.556
Other metabolites
Putrescine	174	1427	–0.0014	–0.0003	0.063	0.045	0.037	0.73	0.59	0.009
Spermidine	174	1952	–0.0010	–0.0004	0.043	0.043	0.024	1.01	0.56	0.320
Uracil	241	1021	0.0001	–0.0004	0.021	0.009	0.015	0.45	0.73	0.619
Urea	147	922	0.0009	–0.0023	0.013	0.034	0.035	2.63	2.75	0.488
Adenine	264	1555	–0.0014	0.0010	0.019	0.025	0.013	1.32	0.65	0.440
Adenosine	236	2342	0.0004	0.0066	0.028	0.111	0.053	3.96 a	1.88 b	0.007
Norvaline	72	783	0.0004	0.0008	0.000	0.006	0.007	Dark sp.	Dark sp.	0.246
Loganin	361	2539	0.0032	0.0032	0.254	0.290	0.287	1.14	1.13	0.557
Thymine	255	1057	0.0003	0.0004	0.011	0.024	0.015	2.14	1.35	0.235
l-Norleucine	158	960	0.0206	0.0045	0.014	0.163	0.226	11.39	15.79	0.040
Porphine	285	1020	0.0013	0.0012	0.008	0.024	0.028	2.91	3.38	0.146
Trans-3-hydroxy-l-proline	230	1221	0.0002	–0.0001	0.000	0.001	0.002	Dark sp.	Dark sp.	0.186
Methyl-β-d-galactopyranoside	204	1699	–0.0177	–0.0082	0.482	0.258	0.229	0.54	0.48	<0.001
N-methyl-d-glutamic acid	260	1382	–0.0086	–0.0006	0.110	0.197	0.033	1.78 a	0.30 b	0.009
1-Methyl-nicotinamide	179	1155	–0.0009	–0.0004	0.015	0.005	0.002	0.31	0.11	0.011

For each identified metabolite, GC-MS characteristics (fragment mass, RI, retention index), loadings in principal components of Fig. 3, average response (resulting from five biological replications), and response ratios of each dark period to the normal photoperiod are presented.

Response ratios representing significant differences (P < 0.05) are indicated in bold, while significant differences between the relative responses measured after 24h and 72h darkness are indicated by different letters. The P-value represents the level of significance between the relative responses of each compound (ANOVA), when at least two average responses are >0.