Comparative study of leaf nutrient reabsorption by two different ecotypes of wild soybean under low-nitrogen stress

Introduction

The identification and evaluation of germplasm resources is a basic and vital challenge for industrial crop improvement (Abdurakhmonov & Abdusattor, 2008). Wild soybean (Glycine soja) is considered the progenitor of cultivated soybean (Glycine max) and has been challenged by the natural environment for thousands of years to withstand various biotic and abiotic stresses (Zhang et al., 2017). Wild soybean maintains a much higher level of genetic diversity than cultivated soybean and represents a treasured genetic resource (Xie et al., 2019). Wild soybean has evolved many eco types under the action of divergent evolution, such as tolerances to salt alkali, drought, barren soil (such as phosphorus or nitrogen deficiency), and so on (Sun et al., 2021). To provide an important theoretical basis for the evaluation of plant resources, it is necessary to explore the mechanism of these exceptional traits in wild soybean (Chen et al., 2019).

Nitrogen (N) is the most important factor limiting plant growth and development, especially at the seedling stage (Luca et al., 2019). The symbiotic nitrogen fixation system of leguminous plants, such as soybean, is not fully established at the seedling stage, and the seedings cannot or rarely use the nitrogen supplied by the symbiotic nitrogen fixation of rhizobia (Mourtzinis et al., 2018). Cultivating low nitrogen (LN) tolerant crop varieties can solve the problem of environmental pollution caused by the application of excessive nitrogen fertilizer in global agricultural production (Arun et al., 2016; Mourtzinis et al., 2018). Wild soybean has high levels of stress tolerance and is an important germplasm resource for improving the stress tolerance of cultivated soybean. It has been reported that, under low-nitrogen stress in the leaves of wild soybean seedlings, the concentration of carotenoids, which can be used as a photo-protective substance to inhibit the increase of reactive oxygen species (ROS) (Zhao et al., 2019). Wild soybean could maintain the balance of carbon and nitrogen metabolism by regulating the activities of nitrogen metabolism-related enzymes to improve the ability to tolerate low-nitrogen stress (Wang et al., 2019). Therefore, it is necessary to improve the LN tolerance of soybean by using appropriate wild germplasm resources.

In recent years, metabolomics and its correlation with ion concentrations had been widely used to determine responses to various abiotic stresses, including salinity, drought, and nutritional deficits (Li et al., 2017; Jiao et al., 2018). Metabolomics provides insights into the deep relationship between metabolites and changes in plant physiological conditions by combining a range of different analytical technologies and calculation methods (Yi et al., 2016). Under nutrient deficiencies, plant autophagic systems and vacuolar proteinases can degrade nitrogen-based macromolecular substances into amino acids, peptides, and urea, and then transport them to nitrogen-demanding organs (Galloway et al., 2004). Metabolomics analysis showed that wild soybean could improve its ability to tolerate nitrogen stress by increasing rates of secondary metabolism, carbohydrate metabolism and amino acid metabolism in the roots (Sun et al., 2021). During senescence of plant leaves, the catabolic processes of macromolecular compounds increase, whereas anabolic processes decrease, and the mobilization and recycling of nutrients occur from old leaves to sinks, such as young leaves, especially under conditions of nutrient deficiency, similar findings have been reported in Arabidopsis thaliana (Mei & Thimann, 1984).

The transfer of some nutrients from senescent tissues to other, more demanding tissues in plants is called nutrient reabsorption (Kichey et al., 2007; Elger et al., 2009). This process can improve the utilization efficiency of nutrients by plants and reduce the dependence on soil nutrients (Taylor et al., 2010; Wei et al., 2018). Nutrient reabsorption is an important mechanism for plants to improve their ability to survive under adversity. Amino acids are important metabolites and carriers of organic nitrogen between plant organs (Häusler, Ludewig & Krueger, 2014). Under stress, amino acids are subsequently recycled and allocated for the synthesis of specific proteins required under nutrient limitation (Araújo et al., 2011). Whereas during carbon or nitrogen starvation, proteins are degraded, and the complete oxidation of their amino acids produces the energy required to fuel the particular needs of stressed leaves. Furthermore, nitrogen uptake rates by roots of intact soybean plants are stimulated by malate, which moves down the phloem and accumulates in the root (López-Bucio et al., 2000). Understanding the relationship between organic acid metabolism and nitrate uptake and utilization could be an important avenue to understand the survival strategies of barren-tolerant wild soybean. The combined analysis of physiological and metabolite profiles has provided a new perspective with which to reveal the reuse of nitrogen between young and old leaves of legumes under LN conditions (Tegeder & Masclaux-Daubresse, 2018).

In the current study, to analyze the tolerance strategies used by wild soybean ecotypes adapted to LN stress, common wild soybean (GS1) and barren-tolerant wild soybean (GS2) were used as the study materials. LN stress was simulated using Hoagland’s nutrient solution containing one-quarter of the non-limiting nitrogen concentration. The photosynthetic rate, ion concentration changes, and the types, quantities, and pathways of small molecular organic metabolites in young and old leaves of the two accessions were determined and analyzed under LN stress. By comparing the differences in leaf nutrient reabsorption between the two ecotypes of wild soybean under LN stress, the mechanism of LN tolerance employed by wild soybean which evolved under divergent adaptation conditions are discussed from the perspective of nutrient reabsorption. The present study provides a quantitative standard by which to select, evaluate and utilize wild soybean germplasm accessions and provides an important scientific basis for research on plant divergent evolution.

Materials and Methods

Results

Photosynthesis characters

Compared with the corresponding plants under CK conditions, P_N decreased significantly 0.64-fold (p < 0.05) and 0.26-fold (p > 0.05) in the young leaves of GS1 and GS2 respectively. C_i/C_a increased significantly 0.07-fold (p < 0.05) and 0.09-fold (p < 0.05) in the young leaves of GS1 and GS2, respectively, and increased 0.05-fold (p < 0.05) and 0.07-fold (p < 0.05) in the old leaves of GS1 and GS2, respectively. Chl(a+b) decreased significantly in the old leaves of GS1 and GS2, especially in GS1 significantly decreased (p < 0.05) (Fig.1).

Response of ion concentrations

Principal component analysis (PCA) was used to differentiate the ion concentrations in the young and old leaves of the two ecotypes of wild soybeans (Fig. 2). The ion concentrations of young leaves in the CK and LN treatments were clearly separated by the first component (PC1), representing 70.9% of the total variation, and NO₃⁻, SO₄²⁻, SO₄²⁻, Cl⁻, B³⁺ and Fe²⁺ were the major contributors (Figs. 2A and 2B; Table S3). PC2 represented 16.9% of the variation between the different ecotypes, of which NO₃⁻, Mn²⁺, H₂PO₄⁻ and Na⁺ were major contributors (Fig. 2A and 2B; Table S3). The CK and LN ion content in old leaves were clearly separated by PC1, representing 58.9% of the total variation, and NO₃⁻, Mg²⁺, Ca²⁺, Cl⁻, B³⁺, and Fe²⁺ were the major contributors (Figs. 2C and 2D; Table S3). PC2 presented a 13.2% variation between GS1 and GS2, and H₂PO₄⁻, SO₄²⁻, C₂O₄²⁻, Mn²⁺, and Na⁺ were major contributors (Figs. 2C and 2D; Table S3).

Under LN stress, NO₃⁻ decreased significantly 0.69-fold and 0.50-fold (p < 0.01) and SO₄²⁻ decreased significantly 0.15-fold and 0.29-fold (p < 0.05) in the young leaves of GS1 and GS2, respectively. Na⁺ and Ca²⁺ concentrations increased significantly 0.34-fold and 0.16-fold (p < 0.05) in the young leaves of GS2. and there was particularly significant enrichment of Mn²⁺ and Zn²⁺ concentrations in the young leaves of GS2. Under LN. The levels of B³⁺, Fe²⁺, H₂PO₄⁻and C₂O₄²⁻ increased significantly in GS1 young leaves (p < 0.05) and highly significantly in GS2 young leaves (p < 0.01). NO₃⁻ concentrations decreased significantly2.10-fold and 1.77-fold (p < 0.01) in the old leaves of GS1 and GS2, respectively, whereas SO₄²⁻ increased 0.37-fold (p < 0.01) in GS1 but decreased 0.98-fold (p < 0.01) in the old leaves of GS2. The concentrations of Cl⁻, H₂PO₄⁻, Mn²⁺, B³⁺, Fe²⁺, and Zn²⁺ increased in the old leaves of GS2 (Table 2).

Table 2:

Ion contents in young and old leaves of two wild soybean ecotypes under LN stress values were presented as the mean ± standard error of four biological replicates.

		GS1		GS2		Fold changes Log2(LN/CK)
		CK	LN	CK	LN	GS1	GS2
Young leaves	Cl−	2.39 ± 0.11	4.47 ± 0.07	2.27 ± 0.17	4.93 ± 0.20	0.90	1.12**
	NO3−	2.26 ± 0.11	1.40 ± 0.01	3.5 ± 0.05	2.48 ± 0.04	−0.69**	−0.50**
	H2PO4−	18.57 ± 0.0.26	31.96 ± 0.97	19.72 ±	23.35 ± 0.31	0.78*	0.24**
	SO42−	26.11 ± 0.20	23.59 ± 0.70	31.5 ± 0.94	25.85 ± 2.23	−0.15*	−0.29**
	C2O42−	1.03 ± 0.06	1.72 ± 0.02	1.58 ± 0.04	1.69 ± 0.08	0.74*	0.10**
	Mn2+	0.09 ± 0.00	0.13 ± 0.00	0.11 ± 0.00	0.20 ± 0.01	0.59*	0.84**
	B3+	0.35 ± 0.01	1.62 ± 0.00	0.53 ± 0.01	1.87 ± 0.07	2.22*	1.81**
	Fe2+	0.05 ± 0.00	0.12 ± 0.00	0.06 ± 0.00	0.13 ± 0.01	1.17*	1.07**
	Zn2+	0.14 ± 0.01	0.24 ± 0.00	0.17 ± 0.01	0.35 ± 0.01	0.78	1.06**
	Na+	2.15 ± 0.15	2.05 ± 0.05	1.64 ± 0.08	2.08 ± 0.15	−0.07	0.34*
	Mg2+	28.30 ± 0.79	26.48 ± 0.13	30.14 ± 0.28	29.57 ± 0.92	−0.10	−0.03
	Ca2+	14.61 ± 0.41	14.00 ± 0.05	13.44 ± 0.07	14.97 ± 0.41	−0.06	0.16*
	K+	191.66 ± 5.18	169.57 ± 0.30	167.2 ± 1.48	174.03 ± 5.27	−0.18*	0.06
Old leaves	Cl−	2.49 ± 0.18	7.77 ± 0.24	1.83 ± 0.08	7.92 ± 0.21	1.64	2.12**
	NO3−	3.96 ± 0.04	0.92 ± 0.04	4.24 ± 0.05	1.24 ± 0.04	−2.10**	−1.77**
	H2PO4−	20.58 ± 0.05	46.04 ± 0.66	16.1 ± 0.45	25.01 ± 0.18	1.16**	0.64**
	SO42−	2.16 ± 0.12	2.79 ± 0.11	55.39 ± 1.34	28.07 ± 0.30	0.37**	−0.98**
	C2O42−	1.33 ± 0.00	1.34 ± 0.04	1.26 ± 0.08	1.14 ± 0.05	0.01	−0.13
	Mn2+	0.12 ± 0.02	0.16 ± 0.04	0.22 ± 0.03	0.3 ± 0.01	0.46	0.43*
	B3+	1.09 ± 0.03	4.86 ± 0.05	1.18 ± 0.13	4.94 ± 0.16	2.16**	2.07**
	Fe2+	0.04 ± 0.00	0.09 ± 0.00	0.06 ± 0.01	0.09 ± 0.01	0.98**	0.68*
	Zn2+	0.09 ± 0.00	0.35 ± 0.00	0.11 ± 0.01	0.29 ± 0.01	1.96	1.35**
	Na+	1.01 ± 0.04	1.82 ± 0.06	1.76 ± 0.11	2.76 ± 0.08	0.85**	0.65**
	Mg2+	46.40 ± 0.01	33.28 ± 0.06	60.62 ± 7.75	42.48 ± 1.55	−0.48**	−0.51
	Ca2+	48.96 ± 0.14	31.32 ± 0.07	58.76 ± 7.66	38.89 ± 0.69	−0.64**	−0.60*
	K+	163.44 ± 0.07	172.59 ± 0.70	166.64 ± 17.11	141.27 ± 3.60	0.08**	−0.24

DOI: 10.7717/peerj.15486/table-2

Note:

GS1, common soybean; GS2, barren tolerant wild soybean; CK, control treatment; LN, low-N stress. * and ** indicate signiûcant (p < 0.05) and highly signiûcant (p < 0.01) diûerences, respectively.

Metabolomics response in young leaves

In PCA, PC1 explained 84% of the variance, in metabolite concentratins changes between the young leaves of GS1 and GS2 (Fig. 3). The main metabolites contributing to PC1 were alanine, 3-aminoisobutyric acid, lactulose, and threitol. The CK and LN-stressed young leaves in GS1 and GS2 were clearly distinguished by PC2, which represented 7.1% of the total variation, with the main factors contributing to PC2 were 3-hydroxypropionic acid, 2-hydroxypyridine, oxalic acid, and maleic acid (Fig. 3A, Table 3 and Table S4). PC3 explained 4.1% of the variance and the main contributors were glucoheptonic acid, threitol, maleic acid, and palmitic acid (Fig. 3A, Table 3 and Table S4). The levels of 63 metabolites changed significantly under LN stress relative to CK in the young leaves of both ecotypes (Table 3).

Table 3:

Changes of LN stress on metabolite content in young leaves of two wild soybean ecotypes.

	Metabolite name	Relative concentration				Fold changes Log2(LN/CK)
		GS1		GS2
		CK	LN	CK	LN	GS1	GS2
Amino acids	Valine	0.62 ± 0.04	0.54 ± 0.05	8.16 ± 0.95	7.05 ± 0.29	−0.19**	−0.21
	Isoleucine	0.39 ± 0.01	0.11 ± 0.00	1.49 ± 0.28	2.76 ± 0.20	−1.83**	0.89*
	Aspartic acid	5.30 ± 0.06	9.35 ± 0.01	7.60 ± 0.51	11.39 ± 0.34	0.82**	0.58**
	Asparagine	2.58 ± 0.67	0.60 ± 0.76	0.11 ± 0.05	0.09 ± 0.01	−2.09**	−0.38
	L-Homoserine	0.22 ± 0.00	0.08 ± 0.00	0.18 ± 0.06	0.99 ± 0.03	−1.56	2.45**
	L-Allothreonine	1.77 ± 0.03	1.82 ± 0.04	2.44 ± 0.15	0.81 ± 0.09	0.04	−1.58**
	Serine	0.19 ± 0.13	0.16 ± 0.25	0.90 ± 0.14	0.68 ± 0.07	−0.27	0.41
	Proline	0.27 ± 0.00	0.21 ± 0.02	0.22 ± 0.02	0.50 ± 0.03	−0.40*	1.21**
	Alanine	0.06 ± 0.04	0.07 ± 0.03	5.05 ± 0.36	24.78 ± 0.75	0.35	2.29**
	Glycine	28.26 ± 0.02	50.61 ± 0.04	6.69 ± 0.57	4.58 ± 0.27	0.84*	−0.54*
	Phenylalanine	11.31 ± 0.07	10.73 ± 0.02	1.89 ± 0.28	0.69 ± 0.13	−0.08	−1.45*
	4-Aminobutyric acid	0.52 ± 0.01	0.32 ± 0.03	2.37 ± 0.12	2.73 ± 0.28	−0.70*	0.21*
	Ethanolamine	55.44 ± 0.02	86.12 ± 0.04	15.31 ± 0.55	8.50 ± 0.35	0.64*	−0.85**
	D-Mannosamine	11.32 ± 0.00	7.19 ± 0.01	0.06 ± 0.05	0.08 ± 0.01	−0.65	0.25
	Sphingosine	1.76 ± 0.30	2.34 ± 0.34	3.08 ± 0.32	2.85 ± 0.54	0.41	−0.11
	3-Aminoisobutyric acid	0.41 ± 0.02	0.23 ± 0.03	0.24 ± 0.02	0.25 ± 0.02	−0.85	0.08
	5-Aminovaleric acid	4.59 ± 0.14	0.80 ± 0.09	0.29 ± 0.07	0.62 ± 0.08	−2.53	1.07*
	2-Hydroxypyridine	0.07 ± 0.00	0.01 ± 0.00	7.20 ± 0.62	7.58 ± 0.25	−2.32	0.07
Sugars and polyols	Sucrose	0.08 ± 0.07	0.07 ± 0.00	1.30 ± 0.17	5.28 ± 0.44	−0.10	2.02**
	Maltose	0.33 ± 0.01	0.28 ± 0.02	0.30 ± 0.04	1.48 ± 0.14	−0.21	2.33**
	Maltotriose	0.93 ± 0.02	0.96 ± 0.03	0.09 ± 0.01	0.05 ± 0.03	0.04	−0.73
	Melezitose	28.10 ± 0.38	20.39 ± 0.02	0.06 ± 0.01	1.29 ± 0.25	−0.46	4.55**
	Threitol	2.57 ± 0.20	1.72 ± 0.08	0.29 ± 0.01	1.17 ± 0.16	−0.58	2.04**
	Galactinol	0.71 ± 0.01	0.52 ± 0.02	0.92 ± 0.09	1.03 ± 0.03	−0.47	0.16
	Glycerol	1.91 ± 0.28	1.56 ± 0.27	8.33 ± 0.27	6.66 ± 0.82	−0.29	−0.32
	Myo-inositol	0.31 ± 0.02	0.22 ± 0.03	43.71 ± 0.55	2.15 ± 0.33	−0.48	−4.35**
	L-Threose	54.13 ± 0.01	60.66 ± 0.16	0.08 ± 0.01	0.02 ± 0.01	0.16	−1.67**
	Xylose	0.09 ± 0.02	0.11 ± 0.01	1.32 ± 0.18	1.37 ± 0.25	0.29	0.05
	Cellobiose	0.98 ± 0.06	0.60 ± 0.02	0.04 ± 0.01	0.31 ± 0.04	−0.72	2.83**
	Lactulose	1.95 ± 0.12	0.05 ± 0.03	0.33 ± 0.04	1.71 ± 0.47	− 5.28	2.39*
	D-Arabitol	0.25 ± 0.13	0.97 ± 0.06	2.28 ± 0.36	1.18 ± 0.09	1.96	0.95*
	Glucose-6-Phosphate	306.86 ± 0.34	216.33 ± 0.17	0.03 ± 0.00	0.03 ± 0.00	−0.50*	0.33
	Fructose 2,6-Biphosphate	0.08 ± 0.00	0.28 ± 0.01	0.15 ± 0.02	0.07 ± 0.02	1.84**	−1.12*
	Maltose	1.50 ± 0.22	2.40 ± 0.25	0.25 ± 0.02	0.61 ± 0.14	0.68	1.30*
Organic acid	Glycolic acid	8.09 ± 0.33	4.01 ± 0.07	0.12 ± 0.00	0.74 ± 0.19	−1.01	2.67*
	Lactic acid	0.15 ± 0.02	0.16 ± 0.04	2.97 ± 0.38	3.47 ± 0.14	0.09	0.22
	Methylmalonic acid	0.55 ± 0.08	0.06 ± 0.01	0.12 ± 0.00	0.08 ± 0.01	−3.13	−0.57*
	Mucic acid	0.20 ± 0.02	0.13 ± 0.02	1.45 ± 0.16	1.84 ± 0.09	−0.64	0.34
	D-Glyceric acid	0.33 ± 0.55	0.43 ± 0.09	2.52 ± 0.25	1.81 ± 0.52	0.36	−0.47
	Pyruvic acid	0.20 ± 0.55	0.13 ± 0.30	1.93 ± 0.08	4.41 ± 0.50	−0.64*	1.19**
	Succinic acid	0.25 ± 0.02	0.32 ± 0.02	6.97 ± 0.69	6.53 ± 0.48	0.37*	−0.09
	Fumaric acid	4.50 ± 0.25	2.40 ± 0.16	3.08 ± 0.04	25.36 ± 1.99	−0.91*	3.04**
	Citramalic acid	7.06 ± 0.3	1.09 ± 0.02	0.45 ± 0.02	0.18 ± 0.04	−2.70**	−1.31**
	Oxalic acid	0.97 ± 1.79	0.78 ± 0.03	0.24 ± 0.02	0.17 ± 0.03	−0.31	−0.49
	Itaconic acid	2.90 ± 0.03	3.91 ± 1.79	3.07 ± 0.35	2.33 ± 0.34	0.43	−0.40
	Malonic acid	4.47 ± 0.08	5.48 ± 0.51	1.12 ± 0.06	9.71 ± 0.55	0.30	3.12**
	Maleic acid	0.08 ± 0.03	0.18 ± 0.03	6.99 ± 0.39	9.24 ± 0.90	1.11*	0.40
	Gluconic acid	0.56 ± 0.07	0.78 ± 0.09	0.07 ± 0.00	0.08 ± 0.02	0.49**	0.16
	Nicotinic acid	4.95 ± 0.58	10.38 ± 0.40	0.69 ± 0.04	1.25 ± 0.15	1.07**	0.85*
	Threonic acid	2.23 ± 0.83	0.72 ± 0.00	1.44 ± 0.21	0.81 ± 0.14	−1.63*	−0.84
	Galactonic acid	0.83 ± 0.00	0.51 ± 0.10	1.01 ± 0.04	1.12 ± 0.08	−0.70**	0.15
	Glucoheptonic acid	16.23 ± 0.31	8.81 ± 0.41	0.22 ± 0.03	0.56 ± 0.02	−0.88	1.37**
	3-Hydroxypropionic acid	0.04 ± 0.00	0.04 ± 0.00	0.19 ± 0.01	0.14 ± 0.01	−0.14	−0.40
	Shikimic acid	0.01 ± 0.00	0.00 ± 0.00	3.90 ± 0.21	1.92 ± 0.49	−1.87	−1.02*
	Stearic acid	0.24 ± 0.01	0.11 ± 0.03	0.24 ± 0.02	0.30 ± 0.03	−1.18*	0.35
	Palmitic acid	42.32 ± 1.77	40.01 ± 1.53	0.32 ± 0.06	0.44 ± 0.14	−0.08	0.48
	Linolenic acid	0.17 ± 0.05	0.14 ± 0.02	1.53 ± 0.12	0.18 ± 0.02	−0.29	−3.06**
	Linoleic acid	4.13 ± 0.09	2.72 ± 0.13	1.82 ± 0.40	2.34 ± 0.19	−0.60**	0.37
	Ferulic acid	13.46 ± 0.03	36.23 ± 0.13	1.58 ± 0.26	0.90 ± 0.12	1.43**	−0.81
	Salicylic acid	0.13 ± 0.34	0.18 ± 0.13	0.39 ± 0.03	0.05 ± 0.02	0.52	−2.94**
	Caffeic acid	1.30 ± 0.00	0.36 ± 0.00	0.02 ± 0.00	0.01 ± 0.00	−1.86*	−0.96
Flavonoids	Neohesperidin	11.03 ± 0.05	7.74 ± 0.21	0.15 ± 0.02	1.02 ± 0.84	−0.51*	2.78
	Prunin	8.47 ± 1.06	13.32 ± 0.36	0.09 ± 0.01	0.23 ± 0.02	0.65**	1.37**

DOI: 10.7717/peerj.15486/table-3

Note:

Values were presented as the mean ± standard error of four biological replicates. GS1, common soybean; GS2, barren tolerant wild soybean; CK, control treatment; LN, low2N stress. * and ** indicate signiûcant (p < 0.05) and highly signiûcant (p < 0.01) diûerences, respectively.

Under LN stress, the concentrations of amino acids increased significantly relative to those of aspartic acid and alanine in young leaves of both ecotypes, whereas the relative concentration of asparagine and valine decreased significantly in the young leaves of GS1 but did not change significantly in those of GS2. The relative concentration of isoleucine, L-homoserine and proline increased significantly 0.89-fold (p < 0.05), 2.45-fold (p < 0.01), and 1.21-fold (p < 0.01), respectively, in the young leaves of GS2 whereas they decreased in those of GS1. The concentration of 4-aminobutyric acid (GABA) content decreased significantly by 0.70-fold (p < 0.05) in the young leaves of GS1 but significantly increased 0.21-fold (p < 0.05) in GS2.

Regarding organic acids metabolites, the relative concentration of pyruvic acid and fumaric acid increased significantly under LN in the young leaves of GS2 but decreased significantly in the young leaves of GS1 compared with the CK. The relative concentration of succinic acid increased significantly 0.37-fold (p < 0.05) in GS1, but no significant change was observed in the young leaves of GS2 under LN stress. The relative concentration of citramalic acid decreased significantly 2.70- and 1.31-fold (p < 0.01) under LN in GS1 and GS2, respectively. The relative concentration of galactonic acid, threonic acid, and caffeic acid decreased significantly 0.70-fold (p < 0.01), 1.63-fold (p < 0.05), and 1.86 -fold (p < 0.05), respectively, under LN in young leaves of GS1, but no significant changes were observed in GS2. Ferulic acid increased significantly 1.43-fold (p < 0.01) in the young leaves of GS1, but no significant changes were observed in the young leaves of GS2. The relative concentration of fatty acids metabolites, including stearic acid and linoleic acid, decreased significantly by 1.18-fold (p < 0.05) and 0.60-fold (p < 0.01) in GS1 under LN, but no significant changes were observed in GS2 (Fig. 4).

Discussion

In the process of adapting to a stressful environment, wild soybean has evolved a large number of ecotypes exhibiting valuable characteristics (Lu et al., 2020). Leaves of different ages respond positively in plants that are tolerating adverse environments (Liu et al., 2019). In the current study, the mechanism of LN tolerance in a wild soybean ecotype was analyzed from the perspective of leaf nutrient reabsorption and we assessed the plants for morphometry and rhizobium formation under LN conditions. N deficiency inhibited the growth of the seedlings of both wild soybean ecotypes according to their different adaptation to LN stress. The biomass of the tolerant GS2 seedlings decreased less than the GS1 seedlings under LN conditions and physiological and metabolite regulation in GS2 may play an important role in the process of tolerating low-nitrogen stress.

In the present study, plant phenotypes and biomass accumulation further confirmed that LN stress could delay plant development, with GS2 exhibiting a greater LN tolerance. TC and TN data display a worse N deficiency in the young leaves of GS1than in GS2. N deficiency will destroy the chloroplast membrane and reduce chlorophyll content (Ohwaki & Sugahara, 1997). Under LN stress, photosynthetic pigment concentrations declined less in the young and old leaves of LN- tolerant GS2 than in GS1, compared with the CK. This helped GS2 to maintain relatively stable photosynthesis rates in both young and old leaves (Chen et al., 2019). The gas-exchange coefficient increased in both ecotypes, with the difference being more significant in the young leaves of GS2 than in GS1, compared with CK. The wild soybean younger leaves have higher P_N and g_s than old leaves, indicating that under low nitrogen stress, the carbon metabolism ability in the new leaves of wild soybean is stronger than that of the old leaves, which is a manifestation of its active resistance to low nitrogen stress. Indicating that GS2 slows down the photosynthetic rate in old leaves to reduce energy and material consumption to maintain relatively stable photosynthetic characteristics and resist LN stress. However, cultivated soybean younger leaves have lower P_N and g_s than old leaves, indicating an imbalance in their photosynthetic characteristics under low nitrogen stress.

It could be seen from the results that NO₃⁻ deficiency in GS1 leaves was more severe than in GS2 leaves which indicated that the barren-tolerant GS2 ecotype was more efficient in the process of absorption and storage of N under LN. The SO₄²⁻ concentrations decreased significantly in the old leaves of GS2 but increased in those of GS1. SO₄²⁻ concentrations decreased more sharply in the young leaves of GS1 than in those of GS2. Sulfur is essential element in the active center of enzymatic reactions (Gao, Zhang & Ma, 2009) and it also plays a certain role in the process of plant growth regulation, detoxification, defense, and stress tolerance (Bollig et al., 2019). Organic sulfur can be converted into inorganic sulfur (mainly sulfate, SO₄²⁻) by protein hydrolysis, sulfate can be converted into organic sulfur by metabolism, and can be transferred to young tissues for reuse (Liu et al., 2020). The results indicate that GS2 transferred most of the sulfate from senescent older leaves to young tender tissues to alleviate the obstruction by ion imbalance of plant growth. Mineral elements have a marked effect on the improvement of nitrogenase activity in the processes of nitrogen reduction and fixation (Lecourt et al., 2015). Previous studies have suggested that changes in the availability of one element could exert an effect on the uptake and accumulation of other elements in plants (Xue et al., 2012). In GS2 the concentrations of Mn²⁺ and Zn²⁺ in the young leaves, and Cl⁻, H₂PO₄⁻, Mn²⁺, B³⁺, Fe²⁺, and Zn²⁺ in the old leaves were particularly significant increased. Indicating beneficial mineral ions were enriched in the old leaves of GS2, which could help GS2 to avoid ion imbalance under LN stress (Zerche et al., 2016).

According to the results of the present study, the relative GABA concentrations decreased significantly in young leaves of GS1, although it increased in both the young and old leaves of GS2. GABA acts as an endogenous signaling molecule in plant growth and development and rapidly accumulates in plant tissue in response to several abiotic and biotic stresses (Nayyar et al., 2014; Priya et al., 2019). Firstly, under LN stress, hydroxyls produced by nitrate reduction decreased and the frequency of electron carriers in the electron transport chain decreased, resulting in a significant increase in ROS (Ramesh et al., 2015). GABA plays a crucial role in the scavenging of ROS generated rapidly due to disruption of the intracellular redox equilibrium and hence significantly mitigates oxidative stress-induced damages in plants (Streeter, Lohnes & Fioritto, 2001). Secondly, GABA has long been considered an important link in C and N metabolism since the GABA shunt pathway competes with the respiratory chain for SSADH (Shelp, Bown & McLean, 1999; Kinnersley & Turano, 2000). Glutamic acid, the precursor of GABA, is considered to be the main form of nitrogen accumulated in the roots of plants. Under an adverse environment, plants can make the nitrogen of glutamic acid flow into GABA and proline to regulate the metabolism of nitrogen, which is a key factor in the rapid response of plants to external stimuli (Nam et al., 2015; Shelp et al., 2012). Under salt stress, the accumulation of GABA in Arabidopsis leads to an increase in amino acids and the activity of glutamate dehydrogenase (GAD), increasing the expression of GAD instantaneously, and thus increasing the flux of the GABA shunt and other related pathways to regulate TC/TN balance (Renault et al., 2010). Under stress, the GABA shunt may perform a cataplerotic role by providing carbon to the cycle through the catabolism of GABA to succinic acid (Barbosa et al., 2010). Moreover, GABA is also a temporary N storage compound. The glutamate to GABA conversion is increased under conditions that inhibit glutamine synthesis, reduce protein synthesis, or enhance degradation (Sita & Kumar, 2020). Evidence also indicates that glutamine and GABA are produced during protein storage and mobilization as a means of recycling Arg-derived N and C (Shelp, Bown & McLean, 1999). Thus, the GABA shunt may be of considerable importance in the N economy of plants. Because, in the current study, the accumulation of GABA in the young and old leaves of GS2 was highly consistent, it can be inferred that GS2 can regulate TC/TN balance, antioxidants concentration, and N storage by enriching GABA content to alleviate stress (Kinnersley & Turano, 2016).

Asparagine has a higher N/C ratio than glutamine and can be used as a long-range transport and storage compound, especially in legumes (Masclaux-Daubresse et al., 2010; Lu et al., 2013). Under abiotic stress, the energy supply is insufficient, GS and Fd-GOGAT are inhibited, whereas AS is activated, and N assimilation proceeded toward asparagine metabolism. In the present study, the relative concentrations of asparagine increased in the old leaves of both GS1 and GS2 and decreased significantly in young leaves of only GS1, which indicates that N was transported and reused in GS2 but does not change significantly in young leaves of GS2. Earlier study had confirmed that increases in asparagine concentrations suggest that it will be easier to render available N for remobilization from senescing leaves (Tegeder & Masclaux-Daubresse, 2018). In the current study, the relative concentrations of L-homoserine and isoleucine increased significantly in young and old leaves of GS2. The effective transport and utilization of these key amino acids between young and old leaves provide a relatively stable material basis for protein synthesis in the vigorous young growth parts of GS2.

Organic acid metabolism is of fundamental importance at the cellular level for several biochemical processes including energy production, formation of precursors for amino-acid biosynthesis, and, at the whole plant level, modulation of adaptation to the environment (López-Bucio et al., 2000). Organic acids exuded by roots originate from the downward transport of organic acids in leaves (Hildebrandt et al., 2015). The present results showed that most differential organic acids (e.g., L-lactic acid, maleic acid, caffeic acid, galactaric acid, benzoic acid, and glyceric acid) increased significantly in the old leaves of GS2 but no significant change was found in the corresponding leaves of GS1. Organic acid metabolism not only provides carbon skeletons during N assimilation but also plays potential roles in osmotic regulation, cation balance, nutrient deficiency-related coping mechanisms, and plant–microbe interactions at the root–soil interface (Gregersen et al., 2013). Organic acids have also been found to stimulate nitrate uptake. Nitrate uptake rates by roots of intact soybean plants are stimulated by malate, which moves down the phloem and accumulates in the root (Touraine, Muller & Grignon, 1992). Some plants have specialized roots that exude large amounts of organic acids (corresponding to up to 23% of net photosynthesis), which acidify the soil and chelate metal ions around the roots, resulting in the mobilization of micronutrients (Schachtman & Shin, 2007).

Fritz et al. (2006) found that the levels of 2-oxoglutarate and other TCA cycle intermediates, including citrate, isocitrate, succinate, fumarate, and malate, which are key regulators of carbon–nitrogen interaction, were lower under LN conditions. In the present study, the results showed that, under LN conditions, most differential metabolites related to glycolysis and the TCA pathway did not change significantly in the young leaves of GS2 under LN stress. However, compared with CK, the differences in concentrations of metabolites associated with the energy consumption stage of glycolysis (fructose-6-phosphate) increased significantly in the young leaves of GS1, under LN stress. In the second stage of glycolysis (energy generation), the relative concentrations of organic metabolites of pyruvic acid decreased significantly in the young leaves of GS1 (Geigenberger et al., 1993). In the old leaves of GS2, in particular, the relative concentrations of glycolysis-related metabolites of fructose-6-phosphate and pyruvic acid increased significantly, indicating that the old leaves of GS2 strengthen and generate energy (Avila-Ospina, Clément & Masclaux-Daubresse, 2017). The increase of L-malic acid and succinic acid concentrations enhanced the TCA cycle in old leaves of GS2. Compared with GS1, the strategies of glycolysis and TCA in the young leaves of GS2 could conserve energy from old leaves and maintain a relatively stable state under LN stress (Huang et al., 2008).

Conclusions

Improved carbon assimilation capacity and maintenance of adequate reabsorption of mineral and organic nutrients is an important survival strategy for barren-tolerant wild soybeans under low nitrogen stress. Compared with the common wild soybean, the barren-tolerant wild soybean has a relatively stable ability to maintain photosynthetic assimilation by young leaves and the supply of inorganic nutrients under low-nitrogen stress. In addition, the enrichment of specific nitrogen-containing compounds in old leaves of GS2, such as GABA and asparagine, can play a substantial role in N storage, TC/TN balance and antioxidant defense and can act as signaling molecules to help barren-tolerant soybean tolerate LN stress. The increase in the relative organic acid concentration in the old leaves of GS2 could improve the utilization rate of N in the soil. The rate of fatty acid catabolism and the TCA cycle in the old leaves of GS2 maintained a relatively high level of energy supply, provided an energy basis for nutrient transport and reuse, and improved the ability to tolerate low-nitrogen stress. Our study provides a quantitative standard by which to select, evaluate and utilize wild soybean germplasm accessions and provides an important scientific basis for research on plant divergent evolution.

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