Introduction

Nitrogen (N) is one of the most important biological elements for plants because it is a component of amino acids, proteins, genetic materials, pigments, and other key organic molecules1,2,3. A shortage of N results in a marked decrease in plant photosynthesis in many crops, and the leaf N content has a good correlation with the photosynthetic capacity4 because up to 75% of leaf N is present in the chloroplasts, with most of it in the photosynthetic apparatus5. The photosynthetic N-use efficiency (PNUE, the ratio of the photosynthetic capacity to the leaf N) is frequently used as an important leaf trait for characterizing leaf photosynthetic economics, physiology and strategy6. Many researchers have attempted to improve our understanding of the inherent variation in PNUE under soil N deficiency1,7,8.

Mesophyll conductance to CO2 and N allocation in the photosynthetic apparatus of a leaf cell are important factors that explain the differences in the PNUE9,10. Mesophyll conductance affects the CO2 contents of the carboxylation site, thus influencing the photosynthetic capacity and PNUE11,12. The N used in the photosynthetic apparatus could be divided into three parts, namely Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase), bioenergetics, and light-harvesting components13. Rubisco is involved in carbon reduction reactions, and it is the most abundant enzyme in photosynthesis14,15. N is invested in bioenergetics, limiting the capacity for electron transport and photophosphorylation, and N is also invested in the contents of chlorophyll a/b protein complexes associated with photosystems I (PSI) and II (PSII), influencing light harvesting13.

Furthermore, N is involved in other components of the leaf cell apart from the photosynthetic apparatus. Cell walls play an important role in the mechanical toughness of plant tissues16 and they accumulate a significant amount of N compounds, at up to 10% of cell wall materials17,18. Trade-offs might occur for N allocation to cell walls versus Rubisco16,18. However, some researchers have suggested that these trade-offs might only be intraspecific19 and present in species lacking leaf N20,21. N is also involved in carbonic anhydrases and aquaporins22, with carbonic anhydrases accounting for 0.5–2% of the total soluble leaf protein23. These proteins play a role in mesophyll conductance (gm) by changing the nature of the diffusing molecule24 and facilitating CO2 diffusion through membranes25. Cell walls could account for >50% of the total resistance and a variable proportion of CO2 diffusion in the mesophyll, significantly affecting the variation of the gm26.

Soil N deficiency could affect the leaf N content, photosynthesis, PNUE, gm, and leaf N allocation in many species. Many researchers have found that the Amax′ (light-saturated net CO2 assimilation rate) and Narea (leaf N concentration per area) were decreased in N-deficient soil1,11,12,27. However, the changes in the PNUEs of different species under soil N deficiency were uncertain; the PNUE values increased1,28, decreased27,29, or showed no marked change7 along the N addition gradients. The gm was also usually decreased with soil N deficiency11,12,30. A lower soil N content could result in smaller chloroplasts31, leading to a decreased chloroplast surface area facing the intercellular air spaces32 and an increased distance between the intercellular space and the catalytic site of Rubisco12. Adding N to the soil could improve the leaf N content in the Rubisco, bioenergetics, and light-harvesting components7,33,34,35, but the changes in the proportion of N in these components were unclear1,11.

Dalbergia odorifera, Erythrophleum fordii, Betula alnoides, and Castanopsis hystrix are suitable species for forestation in southern subtropical China, and they have high economic value36,37,38,39. D. odorifera and E. fordii are N-fixing trees and B. alnoides and C. hystrix are non-N-fixing trees. Recent studies have found that Leguminosae trees with a higher Narea did not have a higher Amax′ than other non-N-fixing species40,41. One possible explanation was that the Leguminosae tree species might allocate less N to Rubisco and bioenergetics than nonlegumes, as shown in previous studies40,41,42. However, there is a lack of information on how the leaf N content, leaf N allocation, mesophyll conductance to CO2 and PNUE of N-fixing trees could be affected by a low soil N content43.

In this study, we investigated the PNUE, photosynthesis, leaf N allocation and mesophyll conductance to CO2 in D. odorifera, E. fordii, B. alnoides and C. hystrix seedling leaves that were exposed to different soil N treatments. The objectives of our study were to 1. understand the effects of soil N deficiency on the PNUE, photosynthesis, leaf N allocation, and gm of these trees; and 2. explore the different plant metabolism response modes between N-fixing and non-N-fixing woody species under soil N deficiency. We assumed that the photosynthetic capacity, PNUE and gm of these trees might be reduced under a soil N deficiency, but the N-fixing trees were less affected.

Results

Effects of soil N treatments on A max′, N area, leaf N content per mass (N mass), leaf mass per area (LMA), and PNUE

The seedling leaf Narea and Nmass values were significant higher in D. odorifera and E. fordii than they were in C. hystrix and B. alnoides under all the soil N treatments, and the PNUE was significantly lower in D. odorifera and E. fordii than it was in C. hystrix and B. alnoides (Fig. 1). The higher Narea and Nmass were direct causes of the lower PNUE in the two N-fixing tree seedlings. A significant decrease was observed in the Amax′, Nmass, and PNUE in the D. odorifera, C. hystrix, and B. alnoides seedling leaves under the low N treatments when compared with the high N conditions, and a significant decrease was observed in the Narea in the C. hystrix and B. alnoides seedling leaves (Fig. 1). The Amax′, Nmass, Narea, LMA and PNUE of E. fordii were less affected by the soil N deficiency (for more details, see Supplementary Table S1). The Amax′ had a significantly positive correlation with the Narea in these tree seedling leaves (P < 0.001; Fig. 2), which showed the importance of N on photosynthesis.

Figure 1
figure 1

Light-saturated photosynthesis (Amax′), leaf N content per area (Narea), leaf N content per mass (Nmass), leaf mass per area (LMA), and photosynthetic-N use efficiency (PNUE) in the seedling leaves from the four studied tree species after exposure to different soil nitrogen (N) treatments. The statistical differences between each characteristic of the different species under three N treatments (mean ± SE) are the results of a one-way analysis of variance (ANOVA) (n = 7). The lowercase letters indicate significant differences at the 0.05 level between different N treatments, and the uppercase letters indicate significant differences at the 0.05 level between the species under the same N treatment. Control, high N; MN, medium N; and LN, low N.

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Figure 2
figure 2

Regression analysis of the leaf nitrogen (N) concentration per area (Narea) and light-saturated photosynthesis (Amax′) of the seedling leaves from the four studied tree species. The determination coefficients (R2) and P-values are shown. The lines fitted for N-fixing and non-N-fixing trees are significantly different (P < 0.05) according to the result of a one-way analysis of covariance (ANCOVA) with Amax′ as a dependent variable, whether it could fix N as a fixed factor, and Narea as a covariate

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Effects of soil N treatments on stomatal conductance (g s), g m, CO2 concentration in substomatal cavities (C i), CO2 concentration at the carboxylation site (C c), and C i–C c

The gs, gm, Ci, and Cc in the B. alnoides seedling leaves were higher than they were in the other three species under any soil N treatments, except for the gm under Control, and the Ci–Cc of B. alnoides seedling leaves was lower than that of the other three species, except under Control (Fig. 3). This finding may be related to the fact that B. alnoides is a deciduous tree. The gm and Cc of D. odorifera were significantly lower under LN than Control (−55.5% and −9.7%, respectively), but the Ci–Cc was significantly higher in the LN treatment than under Control (+56.3%). No significant changes were observed in the gs, gm, Ci, Cc, and Ci–Cc between Control and LN for E. fordii. The gs and gm of C. hystrix were significantly lower under LN than Control (−24.3% and −44.4%, respectively), but the Ci and Ci–Cc were significantly higher under LN than Control (+5.6% and +14.8%, respectively). The gm of B. alnoides was significantly lower under LN than Control (−38.0%), but the Ci and Cc were significantly higher under LN than Control (+14.2% and +21.7% Fig. 3). Different species have different response characteristics to the soil N conditions (More details see Supplementary Table S2).

Figure 3
figure 3

Stomatal conductance (gs), mesophyll conductance (gm), CO2 concentration in substomatal cavities (Ci), CO2 concentration at the carboxylation site (Cc), and Ci–Cc in the seedling leaves of the four tree species after exposure to different soil nitrogen (N) treatments. The statistical differences between each characteristic of the different species under the three N treatments (mean ± SE) are the results of a one-way analysis of variance (ANOVA) (n = 7). The CO2 conductance data were measured under light saturated conditions, and the leaf chamber CO2 concentration was 380 μmol mol−1. The lowercase letters indicate significant differences at the 0.05 level between different N treatments, and the uppercase letters indicate significant differences at the 0.05 level between the species under the same N treatment. Control, high N; MN, medium N; and LN, low N.

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Effects of soil N treatments on maximum carboxylation rate (V cmax) and maximum electron transport rate (J max)

The Vcmax values of E. fordii were significantly higher than those of the other three tree species under the Control and MN treatments. The Jmax values of E. fordii were higher than those of the other three tree species only under MN treatment (Fig. 4). No significant difference was observed in the Vcmax and Jmax of the D. odorifera and E. fordii seedling leaves between the different N treatments. The Vcmax and Jmax of C. hystrix in the LN treatments were 30.5 and 38.1% significantly lower than those obtained from the Control treatment, and the Vcmax and Jmax of B. alnoides were 43.7 and 43.7% significantly lower than those obtained under the Control treatment (Fig. 4). The Vcmax and Jmax of the two N-fixing tree seedlings were less affected by the soil N deficiency (More details see Supplementary Table S3).

Figure 4
figure 4

Maximum carboxylation rate (Vcmax) and maximum electron transport rate (Jmax) in the seedling leaves of the four tree species after exposure to different soil nitrogen (N) treatments. The statistical differences between each characteristic of the different species under the three N treatments (mean ± SE) are the results of a one-way analysis of variance (ANOVA) (n = 7). The lowercase letters indicate significant differences at the 0.05 level between different N treatments, and the uppercase letters indicate significant differences at the 0.05 level between the species under the same N treatment. Control, high N; MN, medium N; and LN, low N.

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Effects of soil N treatments on leaf N allocation proportion of the Rubisco (P R), bioenergetics (P B), light-harvesting components (P L), photosynthetic system (P P), cell wall (P CW), and other parts (P Other)

The PR, PB, PP , and PCW values of C. hystrix were higher than the corresponding values obtained for the other three species under any soil N treatments (Fig. 5). No significant change was observed in the PR, PB, PL, PP , and POther values of D. odorifera under any N treatment; the PCW of D. odorifera in the LN treatment was 71.4% higher than that in the Control treatment. No significant change was observed in the PR, PB, PP , PCW, and POther values of E. fordii under any N treatments, and the PL of E. fordii was 33.3% higher in the LN treatment than in the Control treatment. The LN treatment significantly decreased the PB (−28.6%) and POther (−41.2%), and it increased the PCW (+66.7%) of C. hystrix when compared with the corresponding values obtained under the Control conditions. The LN treatment significantly decreased the PR (−38.5%), PB (−42.9%), PL (−33.3%), and PP (−34.1%), and it increased the PCW (+33.3%) of B. alnoides (Fig. 5). Overall, the N allocation of the two N-fixing tree seedlings changed little, but there was a large change for the two non-N-fixing tree seedlings (for more details, see Supplementary Table S4).

Figure 5
figure 5

Nitrogen (N) allocation proportion of the Rubisco (PR), bioenergetics (PB), light-harvesting components (PL), photosynthetic system (PP), cell wall (PCW), and other parts (POther) in the seedling leaves of the four tree species following exposure to different soil N treatments. The statistical differences between each characteristic of the different species under three N treatments (mean ± SE) are the results of a one-way analysis of variance (ANOVA) (n = 7). The lowercase letters indicate significant differences at the 0.05 level between different N treatments, and the uppercase letters indicate significant differences at the 0.05 level between the species under the same N treatment. Control, high N; MN, medium N; and LN, low N.

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Relationships between parameters

The PR, PB, and PP values showed a significant positive correlation with the PNUE in these tree seedling leaves (P < 0.01; Fig. 6a,b,d). No significant correlation was observed between the PL and PNUE in these trees (Fig. 6c). Significant positive relationships were observed between the gm and PNUE in these tree seedling leaves (P ≤ 0.001; Fig. 7). The changes in PR, PB, and gm were important physiological factors influencing the PNUE.

Figure 6
figure 6

Regression analysis of nitrogen (N) allocation proportions in the photosynthetic system (PP), light-harvesting components (PL), Rubisco (PR), and bioenergetics (PB) with the photosynthetic N use efficiency (PNUE) in the seedling leaves of the four tree species after exposure to different soil N treatments. The determination coefficients (R2) and P-values are shown. The lines fitted for the N-fixing and non-N-fixing trees are significantly different (P < 0.05) according to the results of a one-way analysis of covariance (ANCOVA) with the PNUE as a dependent variable, whether it could fix nitrogen as a fixed factor, and PP, PR, PB, and PL as covariates.

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Figure 7
figure 7

Regression analysis of gm (mesophyll conductance) with the PNUE (photosynthetic nitrogen [N] use efficiency) in the seedling leaves of four tree species following exposure to different soil N treatments. The determination coefficients (R2) and P-values are shown. The lines fitted for the N-fixing and non-N-fixing trees are significantly different (P < 0.05) according to the results of a one-way analysis of covariance (ANCOVA) with the PNUE as a dependent variable, whether it could fix nitrogen as a fixed factor, and gm as a covariate.

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Significant negative relationships were found between the PCW and gm in D. odorifera, E. fordii, and C. hystrix (P < 0.001; Fig. 8a,c,d); no significant relationships were observed in B. alnoides (Fig. 8b). Significant positive relationships were observed between PCW and Ci–Cc in D. odorifera (P = 0.002; Fig. 9a). Significant negative relationships were noted between the PCW and Ci–Cc in E. fordii (P = 0.004; Fig. 9b), and no significant relationships were observed in C. hystrix and B. alnoides (Fig. 9c,d). The improved PCW in D. odorifera might relate to its thicker cell walls, but in E. fordii, it might relate to the higher cell wall density.

Figure 8
figure 8

Regression analysis of the gm (mesophyll conductance) with the PCW (nitrogen [N] allocation proportion of cell wall) in the seedling leaves of four tree species under exposure to different soil N treatments. The determination coefficients (R2) and P-values are shown.

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Figure 9
figure 9

Regression analysis of Ci–Cc (the difference between the CO2 concentration in the substomatal cavities [Ci] and carboxylation site ([Cc]) with the PCW (nitrogen [N] allocation proportion of cell wall) in the seedling leaves of the four tree species under exposure to different soil N treatments. The determination coefficients (R2) and P-values are shown.

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No significant relationships were observed between the PCW and PR in D. odorifera and E. fordii, but significant negative relationships were observed in B. alnoides and C. hystrix (P ≤ 0.002). The cell wall N might influence the variation in N in the Rubisco, thus influencing the photosynthetic capacity in these two non-N-fixing tree seedlings. A regression analysis of the PCW with PR in the B. alnoides seedling leaves under the LN treatment was obtained within the shaded zone. Most Control and MN treatment parameters for B. alnoides and C. hystrix were in the shaded zone, and D. odorifera and E. fordii were found under the shaded zone (Fig. 10). Low soil N increased the competition between the Rubisco and cell wall N.

Figure 10
figure 10

Regression analysis of the PR (nitrogen [N] allocation proportion of Rubisco) and PCW (N allocation proportion of cell wall) in the seedling leaves of the four tree species after exposure to different soil N treatments. The determination coefficients (R2) and P-values are shown. The shaded zone represents the distribution area of the PCW and PR in the presence of the trade-off20.

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Discussion

The leaf N contents of two non-N-fixing tree seedlings, B. alnoides and C. hystrix, were significantly affected by the soil N content (Fig. 1, Supplementary Table S5), which was consistent with previously published studies1,11,12,27. However, the leaf N content of E. fordii was not significantly affected by the soil N content. This finding might be due to its strong N fixation capacity and its maintenance of the N content stability in leaves. Different N treatments significantly affected the Nmass of D. odorifera seedling leaves, but the Narea of D. odorifera was not affected by the soil N content (Fig. 1). Because the Narea was influenced by the Nmass and LMA, the LMA of D. odorifera changed with the soil N gradient (Fig. 1); the maintenance of the Narea at a steady state showed good leaf morphological plasticity. The low soil N content decreased the Amax′ in D. odorifera, B. alnoides, and C. hystrix (Fig. 1) for different reasons. In D. odorifera, the low soil N content primarily decreased its Cc (Fig. 3), which is one of the important raw materials for photosynthesis44, and the CO2 partial pressure is important for Rubisco activity because O2 is a competitive inhibitor of the C assimilatory reaction of Rubisco for promoting the Rubisco oxidation reaction12. For the two non-N-fixing tree seedlings, the low soil N content decreased their Vcmax and Jmax values (Fig. 4), which are the key biochemical parameters of the photosynthetic capacity14,45.

The fraction of the total leaf N allocated to the photosynthetic apparatus46, especially to Rubisco and bioenergetics, could influence the variation in the PNUE1,3,16. The gm could also influence the PNUE32,47 by affecting the Cc11,12. In this study, the PR and PB showed a significant positive correlation with the PNUE (P < 0.001, Fig. 6a,b), and the gm significantly affected the PNUE in the seedling leaves of the four studied tree species (Fig. 7), although the effect of the gm on the PNUE was different among the species48. The LN treatment significantly decreased the gm in D. odorifera and the PR, PB, and gm in B. alnoides (Figs 1 and 5), leading to lower PNUEs in the LN treatment. It has been reported that low soil N could decrease the gm12,49 and N allocation3,29. However, Chen et al. (2014) found an improvement in the PR and PB of female Populus cathayana with improved soil N, but the PR and PB of the males decreased1. Warren (2004) also found that an improvement in the soil N could decrease the PR in Eucalyptus globulus. Some plants might have a different strategy for adapting to the soil N11.

The PNUEs of the two non-N-fixing tree seedlings were significantly higher than those of the two N-fixing tree seedlings under any soil treatment (Fig. 1, Supplementary Table S5), which was first attributed to their relatively low Narea and Nmass (Figs 1, 2, Table S7). The N-fixing species, which could gain N from air through legume bacteria, usually have a higher leaf N content than the non-N-fixing species43,50. High PR and PB (Fig. 4, Supplementary Table S5) were the primary biochemical factors leading to their higher PNUEs. These results were also consistent with other studies40,41,42. The leaves are the photosynthetic organs of plants, and plants have roughly two survival strategies, namely, quick investment-return and slow investment-return51. Two N-fixing trees might belong to the slow investment-return species and use a different strategy to use N, such as compensation for their low productivity through a long leaf lifespan18 and storing N for other processes, such as reproduction1. Two N-fixing tree seedlings might grow well in N-deficient soil, and applying N could increase the growth rates of the two non-N-fixing tree seedlings and promote the growth of artificial forests. Of course, some N-fixing trees have the same N utilization and distribution strategies as non-N-fixing trees, such as Acacia mangium52.

A decrease was observed in the gm of the D. odorifera, C. hystrix, and B. alnoides seedlings under the LN treatment, but the reasons for this decline were different. The changes in Amax′ or Ci–Cc could influence the value of gm. In these tree seedlings, the Amax′ decreased under the LN treatment, but the changes in the Ci–Cc were different. D. odorifera and C. hystrix showed an increased Ci–Cc in the LN treatment, but B. alnoides showed no change in its Ci–Cc value (Fig. 3). After entering through the stomata, the CO2 diffuses through air spaces, cell walls, cytosol, and chloroplast envelopes and finally reaches the chloroplast stroma, where it is fixed by Rubisco26,53. Generally, cell walls account for >50% of the total cell CO2 diffusion resistance and a variable proportion of respiration26. D. odorifera, C. hystrix, and B. alnoides showed improved PCW values in the LN treatment (Fig. 5). Mu et al. (2016) also found an increase in the PCW of maize growing under low-N stress29. D. odorifera showed no significant reduction in its Narea in the LN treatment, and thus there was an increase in the N contents in the cell wall (QCWarea) of D. odorifera (+62.4%, Supplementary Table S6). The percentage of N in the cell wall showed a slight variation in the same species16. An improvement in the NCW of D. odorifera under the LN treatment indicates the high dry mass of the cell wall, resulting in improved LMA16,54, and it might improve the thickness of the cell wall, thereby improving its Ci–Cc value16. However, B. alnoides and C. hystrix showed a reduction in their Narea values in the LN treatment, leading to a smaller change in the QCWarea (+5.9% and +29.6%, respectively, Supplementary Table S6). Thus, there were no significant changes in their LMA and Ci–Cc values. An improvement in the PCW of D. odorifera therefore significantly decreased its Ci–Cc and gm, and no significant relationship was observed between the PCW and Ci–Cc in B. alnoides and C. hystrix (Figs 8, 9).

The PCW did not influence the variation in the Ci–Cc, but it showed a significant negative correlation with the gm in two non-N-fixing trees (Fig. 8). The cell wall N might influence the N variation in Rubisco, thus influencing the Vcmax and Amax′ values. Onoda et al. (2004) and Takashima et al. (2004) observed a trade-off between the cell wall and Rubisco N in Polygonum cuspidatum and in Quercus species, respectively16,18. Zhang et al. (2016) also found this trade-off in Mikania micrantha and Chromolaena odorata28. Hikosaka and Shigeno (2009) considered this relationship unlikely to hold as a general rule; the allocation of N to the cell walls did not explain the variation in the Rubisco19. Harrison et al. (2009) and Qing et al. (2012) believed that this relationship might occur during N leaf deficiency20,21. B. alnoides and C. hystrix showed high PR and PCW values (Fig. 5), and a part of the distribution area in or on the shade zone (Fig. 10; for a further explanation of the shade zone, please see Harrison et al.20) indicates that the free amino acid, NO3−, and NH4+ in the leaves were not sufficient (appearing as low POther) to supply N to both Rubisco and the cell wall20, which explained the existence of a trade-off between the PR and PCW (Fig. 10). It is important to note that the regression analysis of the PCW with the PR in the B. alnoides seedling leaves exposed to the LN treatment was found in the shaded zone; most Control and MN treatments of B. alnoides and C. hystrix were in the shaded zone (Fig. 10). Low soil N increased the competition between the Rubisco and cell wall N.

The two non-N-fixing tree seedling leaves showed improved PCW, and the D. odorifera seedling leaves improved both the LMA and PCW values under the LN treatment (Figs 1, 5). The LMA is the product of leaf thickness and density, and it is positively correlated with leaf toughness55 and is a fundamental defensive trait of plants56,57. The cell wall also directly functions as a defense organ58. We observed that the Nmass values of these trees were affected by the soil N content (Fig. 1). Low nutrient availability limits the growth rate of seedlings and might damage the seedlings during the growing season59. The LN treatment might pose a threat to these seedling leaves; thus, plants need to have tougher leaves to survive16, as shown by the relatively high PCW and LMA in D. odorifera seedling leaves and high PCW in the B. alnoides and C. hystrix seedling leaves. Givnish (2002) hypothesized that soil fertility is the primary driver of the leaf lifespan60, and a high LMA leads to a long leaf lifespan51. Therefore, an improvement in the LMA might also increase the leaf lifespan of D. odorifera seedling leaves, ultimately maximizing the carbon assimilation per unit of nutrient over the lifespan of the leaf61,62,63. Different species have different response characteristics to the soil N conditions.

To understand the changes in the various parameters under low soil N in the four species, we drew a process diagram (Fig. 11). Generally, we found fewer parameter changes in the two N-fixing tree seedlings and more parameter changes in the two non-N-fixing tree seedlings. The physiological and ecological characteristics of these two N-fixing tree seedlings are more stable, and these two N-fixing tree seedlings could be good tree species for afforestation in N-poor areas. We also performed Between-Subjects effects tests on the tree varieties and N treatments for the variables in the four species (Supplementary Table S8). In general, varieties of the trees were more important than the N treatment interaction effect, but the N treatment interaction effect was more important in influencing the Amax′ and gm. More trees and more variables must be further studied.

Figure 11
figure 11

Changes in the variables under low soil nitrogen in four species.

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Conclusions

In revisiting our questions, we concluded that (1) soil N deficiency significantly decreased the leaf N concentration and photosynthesis ability in two non-N-fixing trees, but it had less influence on these indices in the two N-fixing trees. (2) The LN treatment had a lower gm in D. odorifera and had lower PR, PB and gm in B. alnoides, eventually resulting in their low PNUE values. (3) D. odorifera, B. alnoides, and C. hystrix seedling leaves showed improved PCW and (or) LMA to adapt to a low-N soil environment. These findings were important for understanding the ecophysiological changes in plants under low soil N conditions. Our findings suggested that the two N-fixing tree seedlings could grow well in N-deficient soil, and they could be good tree species for the afforestation of N-poor areas. Adding N may increase the growth rates for the two non-N-fixing tree seedlings and promote the growth of artificial forests. Because these species live in the same area, it is possible to mix non-N-fixing with N-fixing tree seedlings for afforestation, and mix N-fixing trees in non-N-fixing pure forest after intermediate cutting or selective cutting in non-N-fixing pure forest, which could improve soil N utilization efficiency.

Materials and Methods

Study area and plant material

This study was performed in the Experimental Center of Tropical Forestry (22°7′19″–22°7′22″N, 106°44′40″–106°44′44″E) at the Chinese Academy of Forestry located in Pingxiang, Guangxi Province, China. This location has a subtropical monsoon climate with distinct dry and wet periods, and the mean annual temperature is 21 °C. The mean monthly minimum and maximum temperatures are 12.1 and 26.3 °C, respectively. The mean annual precipitation, which takes place primarily from April to September, is 1400 mm. The active accumulated temperature above 10 °C is 6000–7600 °C. The total annual sunshine duration is 1419 hours64,65.

The seeds of D. odorifera, E. fordii, and C. hystrix were collected separately from the mother trees, and the B. alnoides seedlings were somaclones. The D. odorifera, E. fordii, and C. hystrix seeds were germinated in a seedbed in February of 2014, and B. alnoides was budding at the same time. When the seedlings were approximately 20 cm tall, 90 similarly sized seedlings per species were transplanted to pots (5.4 L, filled with washed river sand) and established in an open site at the Experimental Center of Tropical Forestry in March, 2014.

From April to June, three levels of soil N treatments were set up (Hyponex M. Scott & Sons, Marysville, OH, USA, dissolved in the water from the aqueous solution preparation). Nitrogen fertilizer was applied ten times, once per week. A total of 0.2 (low nitrogen, LN), 0.7 (medium nitrogen, MN), and 1.5 g (set as Control) of available N were applied per pot, with each treatment including 30 seedlings per species. The forms of N that were applied in this study were mixed N (both NH4+ and NO3−), and the NH4+ to NO3− ratio was 1:1. We chose these forms because we used washed river sand as a culture substrate with a pH value of approximately 7, and only using NH4+ or NO3− might cause the soil to become more acid or alkaline, respectively, affecting the plant growth. Wu et al. (2012) found that the proper amounts of N applications for D. odorifera seedlings were 1.74–2.15 g N per pot66. Li et al. (2003) found that the appropriate N applications for E. fordii seedlings were approximately 1.39–1.86 g N per pot67. Although the purpose of this research is to understand the effects of soil N deficiency on plant metabolism, we also want to explore the plant physiological process from a comparatively sufficient to a lack of soil N, because non-N-fixing woody species might be more sensitive to changes in the soil N gradient, and the different ecophysiological processes between a comparatively sufficient to a lack of soil N could help us to understand the effects of soil N deficiency on plant metabolism. Therefore, we set up a high N treatment as Control. The seedlings in each treatment were watered every day to keep the soil moist. Natural light (100% light in the field) was used for illumination.

Determination of gas exchange parameters

Fifteen days after the last N fertilization, on sunny days from 9:00 to 11:00 h in July and August of 2014, seven healthy and similarly sized seedlings were chosen per treatment, per species. One healthy and mature leaf per seedling that was exposed to the sun was chosen to determine the gas exchange parameters. These parameters were determined with a LiCor-6400 portable photosynthesis system (LI-COR, Lincoln Nebraska, USA), and the photosynthetic response to the photosynthetic photon flux density (PPFD, µmol m−2 s−1) and Ci (μmol mol−1) were determined. Under 380 μmol mol−1 of leaf chamber CO2 concentration (the average air CO2 concentration in the day time), the photosynthetic rates were measured under photon flux densities of 1500, 1200, 1000, 800, 600, 400, 200, 150, 100, 80, 50, 30, 20, 10 and 0 μmol m−2 s−1. Under a saturated PPFD, the photosynthetic rates were detected using the same leaf-under leaf chamber CO2 concentrations of 380, 200, 150, 100, 80, 50, 380, 600, 800, 1000, 1200, 1500, 1800 and 2000 μmol mol–1 28,47. We started at a 380 μmol mol−1 concentration because this is the average air CO2 concentration during the day time that could reduce the plant activation time28. The relative humidity of the air in the leaf chamber was maintained at 60–70%, and the leaf temperature was set to 30 °C. The values for the following data or parameters were determined: the net photosynthetic rate (An, μmol m−2 s−1), Amax′ (μmol m−2 s−1), gs (mol CO2 m−2 s−1), and dark respiration (Rn, μmol m−2 s−1). The light- and CO2-saturated net CO2 assimilation rate (Amax, μmol m−2 s−1) was calculated according to Farquhar et al.14. The relative humidity of the air in the leaf chamber was maintained at 60–70%, and the leaf temperature was set to 30 °C.

Determination of the chlorophyll fluorescence, mesophyll conductance, V cmax, and J max

The fluorescence yield was measured using a LiCor-6400 leaf chamber fluorometer (6400–40, LI-COR, Lincoln, Nebraska, USA) on the same leaf and with seven repetitions for each species. The chamber relative humidity and leaf temperature were controlled under the same conditions as described in the gas exchange parameters. The leaf chamber CO2 concentration was set to 380 μmol mol−1. The fluorescence yield (ΔF/Fm′) was subsequently determined. The photosynthetic electron transport rate (Jf, μmol m−2 s−1) was calculated according to the equation described by Loreto et al.68 as follows:

$${J}_{f}=PPFD\times \frac{{\rm{\Delta }}F}{{F}_{m}^{\prime} }\times Leafreflu\times PARDistPhotosys$$
(1)

where PPFD is the photosynthetic photon flux density; Leafreflu is the leaf absorptance valued between 0.82–0.8569 (we used 0.85 in this paper); and PARDistPhotosys is the fraction of quanta absorbed by photosystem II (valued as 0.5)68. The mesophyll conductance (gm, mol CO2 m−2 s−1) was calculated using three different methods to obtain a more accurate value. The variable J method was described by Harley et al.70, and it has been commonly used in recent years71,72,73. The A-Ci curve fitting method was described by Ethier and Livingston74, and Sharkey et al.75 developed a software package to estimate the gm and other parameters based on this method. The exhaustive dual optimization (EDO) method described by Gu et al.76 could estimate up to eight parameters, including the gm, and we obtained an automated analysis of A-Ci curves through a website (http://www.leafweb.org) by uploading our data to determine the value of the gm. Subsequently, the gm calculated by these three methods was used to calculate Cc (μmol mol−1) as follows:

$${C}_{C}={C}_{i}-\frac{{A}_{max}^{\prime} }{{g}_{m}}$$
(2)

The Cc and gm calculated using the three methods are shown in Supplementary Table S9. The mean value of Cc was used to fit the An-Cc curve, followed by the calculation of Vcmax (μmol m−2 s−1) according to Farquhar et al.14 and the Jmax (μmol m−2 s−1) according to Loustau et al.77. The running fitting model used in the in vivo Rubisco kinetics parameters (i.e., Ko, Kc, and their activation energy) was measured according to Niinemets and Tenhunen13.

Determination of additional leaf traits

After the gas exchange parameters and fluorescence yield were determined, the leaf samples and nearby leaves (30–50 leaves per seedling in total, the sizes of which were similar to those of the leaves used to determine the photosynthesis, healthy and mature characteristics, and sun-exposed parameters) were collected from each pot. The surface areas of 10–20 leaves were measured using a scanner (Perfection v700 Photo, Epson, Nagano-ken, Japan). The leaves were subsequently oven-dried to a constant weight at 80 °C for 48 h. The dry weight was measured using an analytic balance, and then the LMA (g m−2) was calculated. The dried leaf samples were ground into dry flour. The organic carbon (C) concentration was determined by potassium dichromate-sulfuric acid oxidation method (Cmass mg g−1, Supplementary Table S10). The N concentration was determined using a VELP automatic Kjeldahl N determination apparatus (UDK-139, Milano, Italy), and then the Nmass (mg g−1) and Narea (g m−2) values were calculated. Then, PNUE (μmol mol−1 s−1) was calculated using the following formula:

$${\rm{PNUE}}=\frac{{A}_{{\rm{\max }}}^{\prime} }{{N}_{{\rm{area}}}}\times 14$$
(3)

where 14 is the atomic mass of nitrogen.

The remaining 20–30 leaves were frozen and kept for laboratory analysis. The frozen leaves (0.2 g, 5–10 leaves) were cut into small 5–10-mg pieces. The leaves were placed in a volumetric flask and brought to a consistenttant volume of 25 mL using 95% (v/v) alcohol. The volumetric flask was protected from light for 24 h, and then the chlorophyll contents were determined using a Shimadzu ultraviolet-visible spectrophotometer (UV 2250, Fukuoka, Japan). For the chlorophyll contents, please see Supplementary Table S10.

The remaining frozen leaves were used to determine the cell wall N content according to the method of Onoda et al.16 as follows: 1 g of leaves was powdered in liquid N and suspended in sodium phosphate buffer (pH 7.5, 25 mL), the homogenate was centrifuged at 2500 g for 5 min, and the supernatant was discarded. The pellet was washed with 3% (w/v) SDS, amyloglucosidase (35 units ml−1, Rhizopus mold, Sigma, St Louis, USA) and 0.2 M KOH and then heated and centrifuged, and the remaining pellet was washed with distilled water and ethanol and then dried in an oven (75 °C) for 2 days (for more details see Onoda et al.)16. The nitrogen content of the rest of the pellet (cell wall N) was determined using a VELP automatic Kjeldahl N determination apparatus. The PCW represents the ratio of the cell wall N content to the total N content.

Calculation of the N allocation in the photosynthetic apparatus

The N allocation fractions of each component in the photosynthetic apparatus were calculated according to Niinemets and Tenhunen13, which has been widely used in recent years1,45,78.

$${P}_{{\rm{R}}}=\frac{{V}_{{\rm{cmax}}}}{6.25\times {V}_{{\rm{cr}}}\times {\rm{LMA}}\times {N}_{{\rm{mass}}}}$$
(4)
$${P}_{{\rm{B}}}=\frac{{J}_{{\rm{\max }}}}{8.06\times {J}_{{\rm{mc}}}\times {\rm{LMA}}\times {N}_{{\rm{mass}}}}$$
(5)
$${P}_{{\rm{L}}}=\frac{{C}_{{\rm{Chl}}}}{{C}_{{\rm{B}}}\times {N}_{{\rm{mass}}}}$$
(6)

where CChl is the chlorophyll concentration (mmol g−1), Vcr is the specific activity of Rubisco (μmol CO2 g−1 Rubisco s−1), Jmc is the potential rate of photosynthetic electron transport (μmol electrons μmol−1 Cyt f s−1), and CB is the ratio of leaf chlorophyll to leaf N during light-harvesting (mmol Chl (g N)−1). The Vcr, Jmc, and CB were calculated according to Niinemets and Tenhunen13.

Statistical analysis

The differences between the seedling leaves of the four tree species, the N-fixing and non-N-fixing tree seedlings, and the three levels of soil N were analyzed by performing a one-way analysis of variance (ANOVA), and a post-hoc test (Tukey’s test) was conducted to determine if the differences were significant. The effects of the tree varieties and N treatments on the variables in the four species were analyzed by two-way ANOVA and Tukey’s test. The significance of the correlation between each pair of variables was tested with a Pearson’s correlation (two-tailed). All the analyses were performed using the Statistical Product and Service Solutions 17.0 program (SPSS17.0, Chicago, USA).