*2.5. Specific Leaf Area (SLA) and Leaf Dry Matter Content (LDMC)*

N addition, alone, significantly increased the SLA values of the three species, and the low level of N addition alone (N50P0) decreased the LDMC of the two grasses (*p* < 0.05, Figure 6). P addition, alone, significantly increased SLA of *L. davurica*, while it had limited effects on the two grasses (Figure 6). N and P addition interaction significantly affected both the SLA and LDMC values of all three species. Under the addition of N50 combined with P, the SLA of *B. ischaemum* and *L. davurica* increased significantly (*p* < 0.05), and the SLA of *S. bungeana* increased under N50P40, while it decreased significantly under N50P80 (*p* < 0.05); LDMC was comparable between different levels of P additions in the two grasses, but it decreased in *L. davurica*. Under the addition of N100 combined with P, the SLA values of *B. ischaemum* and *L. davurica* increased significantly, and the SLA of *S. bungeana* increased (*p* < 0.05) only under the high level of N addition (N100P80); LDMC, among different levels of P addition (i.e., N100P40 and N100P80), was comparable in *B. ischaemum* and *L. davurica*, while it significantly decreased under N100P80 in *S. bungeana* (Figure 6). *Plants* **2022**, *11*, x FOR PEER REVIEW 8 of 17 the SLA values of *B. ischaemum* and *L. davurica* increased significantly, and the SLA of *S. bungeana* increased (*p* < 0.05) only under the high level of N addition (N100P80); LDMC, among different levels of P addition (i.e., N100P40 and N100P80), was comparable in *B. ischaemum* and *L. davurica*, while it significantly decreased under N100P80 in *S. bungeana*  (Figure 6).

**Figure 6.** Specific leaf area (SLA) and leaf dry matter content (LDMC) of the three species under different N and P addition treatments. Different capital letters above the column indicate significant differences among P additions under each N addition rate, while different small letters indicate significant differences among N additions under each P addition rate. Numbers in parentheses are *F*-values, while '\*', '\*\*', and '\*\*\*' indicate *p* ≤ 0.05, *p* ≤ 0.01, and *p* ≤ 0.001, respectively. 'n.s.' indicates no significant difference. SPAD, and SLA values (Figure 9). **Figure 6.** Specific leaf area (SLA) and leaf dry matter content (LDMC) of the three species under different N and P addition treatments. Different capital letters above the column indicate significant differences among P additions under each N addition rate, while different small letters indicate significant differences among N additions under each P addition rate. Numbers in parentheses are *F*-values, while '\*', '\*\*', and '\*\*\*' indicate *p* ≤ 0.05, *p* ≤ 0.01, and *p* ≤ 0.001, respectively. 'n.s.' indicates no significant difference.

*P*Nmax was positively correlated with WUE, SPAD, *N*mass, and SLA in all three species, while it was negatively correlated with LDMC in *S. bungeana* and *L. davurica* (Figure 7). PCA analysis showed that the variance explained by the first and second principal components was 37.5% and 23.5%, respectively, with a total value of 61% (Figure 8). The first principal component had a high correlation with *P*Nmax, WUE, *P*mass, and SLA; the second *P*Nmax was positively correlated with WUE, SPAD, *N*mass, and SLA in all three species, while it was negatively correlated with LDMC in *S. bungeana* and *L. davurica* (Figure 7). PCA analysis showed that the variance explained by the first and second principal components was 37.5% and 23.5%, respectively, with a total value of 61% (Figure 8). The first principal

principal component had high correlation with LDMC. SPAD, *N*mass, and *Nmass/ Pmass* ratio are correlated with both principal components (Figure 8). In a score plot of PCA analysis,

N addition level, and *S. bungeana* gradually moved to the upper right. Under P addition, alone, *L. davurica* gradually moved to the right with P addition level, and *B. ischaemum* slightly moved to the upper right. Under the addition of N combined with P, with the increase in fertilizer application, all three species moved towards higher *P*Nmax, WUE,

component had a high correlation with *P*Nmax, WUE, *P*mass, and SLA; the second principal component had high correlation with LDMC. SPAD, *N*mass, and *Nmass/ Pmass* ratio are correlated with both principal components (Figure 8). In a score plot of PCA analysis, under N addition alone, *B. ischaemum* and *L. davurica* gradually moved to the right with N addition level, and *S. bungeana* gradually moved to the upper right. Under P addition, alone, *L. davurica* gradually moved to the right with P addition level, and *B. ischaemum* slightly moved to the upper right. Under the addition of N combined with P, with the increase in fertilizer application, all three species moved towards higher *P*Nmax, WUE, SPAD, and SLA values (Figure 9). *Plants* **2022**, *11*, x FOR PEER REVIEW 9 of 17 *Plants* **2022**, *11*, x FOR PEER REVIEW 9 of 17

**Figure 8.** Principal component analysis (PCA) of photosynthetic characteristics (*P*Nmax and WUE) and leaf functional traits (SPAD, *N*mass, *P*mass, SLA, and LDMC) in the three species under N and P additions. (**A**) Loadings for each leaf trait; (**B**) Factor scores for each species. **Figure 8.** Principal component analysis (PCA) of photosynthetic characteristics (*P*Nmax and WUE) and leaf functional traits (SPAD, *N*mass, *P*mass, SLA, and LDMC) in the three species under N and P additions. (**A**) Loadings for each leaf trait; (**B**) Factor scores for each species. **Figure 8.** Principal component analysis (PCA) of photosynthetic characteristics (*P*Nmax and WUE) and leaf functional traits (SPAD, *N*mass, *P*mass, SLA, and LDMC) in the three species under N and P additions. (**A**) Loadings for each leaf trait; (**B**) Factor scores for each species.

**Figure 9.** Score plots of PCA analysis of photosynthetic characteristics (*P*Nmax and WUE) and leaf economic traits (SPAD, Nmass, Pmass, SLA, and LDMC) in the three species under N and P additions. (**A**) N addition alone; (**B**) P addition alone; (**C**) N and P combined addition. **Figure 9.** Score plots of PCA analysis of photosynthetic characteristics (*P*Nmax and WUE) and leaf economic traits (SPAD, Nmass, Pmass, SLA, and LDMC) in the three species under N and P additions. (**A**) N addition alone; (**B**) P addition alone; (**C**) N and P combined addition.

#### **3. Discussion 3. Discussion**

The diurnal dynamics of photosynthesis reflect plants' sustained ability to carry out physiological metabolism and biomass accumulation throughout the daytime [45,46], which have been extensively studied in numerous dryland species (e.g., [19–22]). Our results, corroborated with others, showed that the diurnal course of photosynthetic rate showed a double-peaked curve in the three grassland dominant species, and they showed an evident "noon break" of photosynthesis. The noon break is a mechanism to avoid stresses such as excess light, high temperature, and water deficit during the midday [47], and it is a result of stomatal or non-stomatal restriction [48]. Our measurements showed that, during the period of 10:00–12:00 h, the *P*n values gradually decreased while the *L*<sup>s</sup> values increased (Figure 2 and Table 1), suggesting the "noon break" was likely caused by stomatal limitation [46,48]. In line with our first hypothesis and consistent with others (e.g., [49,50]), the *P*n values of the three species considerably increased by nutrient addition, particularly during the peak photosynthetic period (~10:00 h). This is expected since environmental conditions (e.g., light and temperature) are relatively optimal for photosynthesis during this period, hence the promotion of nutrient addition would be most effective. In addition, during the noon time with high air temperature and light radiation, the *Pn* was slightly increased after fertilization, which may be due to increased stomatal conductance due to N and P addition, and this ostensibly alleviated the "noon break" [51]. Our study indicated that N and P fertilization could improve the photosynthetic ability of the three species at the diurnal scale and increase the daily accumulative carbon assimilation. However, we only focused on short-term responses during the peak growing season (i.e., July), so future studies should be taken to further assess intra- or interannual patterns The diurnal dynamics of photosynthesis reflect plants' sustained ability to carry out physiological metabolism and biomass accumulation throughout the daytime [45,46], which have been extensively studied in numerous dryland species (e.g., [19–22]). Our results, corroborated with others, showed that the diurnal course of photosynthetic rate showed a double-peaked curve in the three grassland dominant species, and they showed an evident "noon break" of photosynthesis. The noon break is a mechanism to avoid stresses such as excess light, high temperature, and water deficit during the midday [47], and it is a result of stomatal or non-stomatal restriction [48]. Our measurements showed that, during the period of 10:00–12:00 h, the *P*<sup>n</sup> values gradually decreased while the *L*<sup>s</sup> values increased (Figure 2 and Table 1), suggesting the "noon break" was likely caused by stomatal limitation [46,48]. In line with our first hypothesis and consistent with others (e.g., [49,50]), the *P*<sup>n</sup> values of the three species considerably increased by nutrient addition, particularly during the peak photosynthetic period (~10:00 h). This is expected since environmental conditions (e.g., light and temperature) are relatively optimal for photosynthesis during this period, hence the promotion of nutrient addition would be most effective. In addition, during the noon time with high air temperature and light radiation, the *P<sup>n</sup>* was slightly increased after fertilization, which may be due to increased stomatal conductance due to N and P addition, and this ostensibly alleviated the "noon break" [51]. Our study indicated that N and P fertilization could improve the photosynthetic ability of the three species at the diurnal scale and increase the daily accumulative carbon assimilation. However, we only focused on short-term responses during the peak growing season (i.e., July), so future studies should be taken to further assess intra- or interannual patterns of their photosynthesis to better understand the long-term effects of fertilization.

of their photosynthesis to better understand the long-term effects of fertilization Both N and P are essential elements of key compounds involved in the photosynthetic process, and appropriate N and P additions would increase the content of these Both N and P are essential elements of key compounds involved in the photosynthetic process, and appropriate N and P additions would increase the content of these compounds and, subsequently, the photosynthetic rate [17,26,30]. This was observed in our study: the

*P*Nmax, SPAD, and *N*mass of the three species increased significantly after N addition alone, and there were strong positive correlations between *N*mass, SPAD, and *P*Nmax (*p <* 0.05; Figures 3–5 and 7). The increase in *P*Nmax and WUE with the N addition level was much greater (larger regression slopes) in the two grass species than in the legume *L. davurica* (Table 2), with the greatest increase (the largest slope) of *P*Nmax and WUE, along with the N addition level, in C<sup>4</sup> grass *B. ischaemum* (Table 2). Together, this suggested that the two grasses were more sensitive to N addition alone than the legume. This is consistent with our previous study quantifying the plant biomass of *B. ischaemum* and *L. davurica* mixtures under varying soil moisture and nutrient supplies [52]. We suspect that the subshrub *L. davurica* may not be N-limited due to its N fixation ability and is, thereby, insensitive to exogenous N fertilization. On the other hand, the photosynthetic rate does not continuously increase with N addition amounts after passing a threshold [29,53], which was also observed, here, as the *P*Nmax values of the three species were not significantly different between N50 and N100 (Figures 3 and 6; Table 1). Fossil fuel combustion and extensive fertilization have greatly increased atmospheric N deposition globally in recent decades [54]. Chronic N input by long-term N deposition may, hence, alleviate N limitation and promote plant photosynthesis and growth of regional grassland species, but it may, meanwhile, intensify plant P limitation by increasing P demand [55,56].

**Table 2.** Regression slopes (SE) derived from the multiple linear regression analysis between photosynthetic characteristics (*P*Nmax and WUE) and N addition, P addition, and N and P addition interaction in the three species.


' n.s.', '\*', '\*\*', and '\*\*\*' indicate *<sup>p</sup>* > 0.05, *<sup>p</sup>* <sup>≤</sup> 0.05, *<sup>p</sup>* <sup>≤</sup> 0.01, and *<sup>p</sup>* <sup>≤</sup> 0.001, respectively. Significant slopes are in bold.

Here, the P addition, alone, had greater effects on *P*Nmax, SPAD, and SLA of the leguminous *L. davurica* among the three species (Figures 3, 4 and 6; Table 2). This may be ascribed to P, as it could promote the activity of nitrogenase in the root nodules of legumes and enhance their photosynthesis [34,57], and elevated leaf P content can also directly improve photosynthetic capacity by promoting ATP and NADPH synthesis, as well as regeneration of RuBP [33,58]. Compared with N or P addition alone, the three species had higher *P*Nmax, WUE, and SLA values under N and P combined additions, suggesting a synergetic effect of N and P on plant photosynthesis (Figures 5 and 6). This confirms our second hypothesis and suggests that appropriate N and P combined fertilization should be considered to maintain regional grassland productivity. A myriad of studies have documented this synergetic effect in grasslands worldwide (e.g., [59,60]). Previous studies in the Loess Plateau grasslands also reported the N and P combination had synergetic effects on community productivity [44]. A recent long-term (over 66 years) nutrient addition study in a mesic grassland in South Africa also highlighted that N and P combined addition promoted plant P acquisition and uptake (e.g., increased organic P storage, P recycling, and plant P utilization), which may contribute to the synergetic effect of N and P combined addition [59].

Drylands (e.g., the semiarid Loess Plateau) are often co-limited by water and nutrients [8], as well as characterized by frequent drought events, which greatly impact plant N and P uptake [61]. Nutrient addition, such as N, at an appropriate rate could improve post-drought recovery of grassland and increase the aboveground biomass production [62]. Contrarily, some studies reported that nutrient addition increased grassland drought sensitivity and constrained its recovery from drought events [10]. Besides, grass species with different photosynthetic pathways (C<sup>3</sup> vs. C4) may respond differentially to drought and rewatering under nutrient addition conditions [63]. For the regional grassland, previous studies have quantified the photosynthetic responses of dominant species following rainfall events and reported species-specific patterns [64]. Nevertheless, the interaction of soil moisture (especially drought) and fertilization on dominant species performance remains less understood in the regional grassland and should be assessed, considering recurrent drought events, under future climate scenarios [65].

Leaf functional traits, particularly those so-called economic traits, are invoked to explain plant resource acquisition and utilization [36]. Among them, the leaf *Nmass/ Pmass* ratio indicates environmental N and P availability where the plant grows [56]. In general, *Nmass/ Pmass* ratio less than 10 indicates the N limitation, and greater than 20 indicates the P limitation [56]. The leaf *Nmass/ Pmass* ratio of the three species, averaged across treatments, was 18.8 (*B. ischaemum*), 11.3 (*S. bungeana*), and 24.3 (*L. davurica*), respectively (Figure 5), suggesting species-specific N and P limitations. The *Nmass/Pmass* ratio of the two grasses increased significantly with N addition, while no noticeable change was found in *L. davurica* (Figure 5). This indicates that N addition may lead to P limitation in the two grass species. Meanwhile, increased soil N and P availability would release plants from nutrient competition to other resource competition, such as light and water [66]. Grassland dominant species may accordingly alter their leaf functional traits to maximize light harvesting to maintain dominance. According to the LES theory, plants with higher light capture, resource acquisition, and turnover capacity show higher SLA, *N*mass, and *P*mass in contrast to the slow-growth ones with higher LDMC and conservative nutrient resource use [36]. Similar to other studies (e.g., [66]), the three species studied here shifted to a fast-growth strategy after N addition with larger, thinner, and N-rich leaves (higher SLA, *N*mass, and SPAD), as well as higher assimilation rate per unit leaf area (higher *P*Nmax). Though score plots from PCA analysis indicated that, under N addition, three species adopted different strategies to improve their light harvesting: C<sup>4</sup> grass *B. ischaemum* mainly by increasing SLA and *P*Nmax, while C<sup>3</sup> grass *S. bungeana* and C<sup>3</sup> subshrub *L. davurica* primarily increased leaf N content and SPAD (Figure 9), and only *L. davurica* had notable shifts in photosynthetic and leaf functional traits under P additions (Figure 9B), which suggests that the three species had different trade-off strategies in photosynthetic performance and leaf economic traits in response to N and/or P addition [17,66], and these should be considered when assessing N and P fertilization effects on community structure and functions. The *P*Nmax values of the three species were mostly highest under the 'N50P400 treatment among all treatments, indicating that it could be considered an optimal fertilization measure for improving grassland production.

#### **4. Materials and Methods**

#### *4.1. Site Description*

This work was conducted at the Zhifanggou watershed (109◦13'46"–109◦16'03" E, 36◦42'42"–36◦46'28" N), located in the Ansai District, Yan'an City, Shaanxi Province, China. It has a semiarid continental monsoon climate. The mean annual temperature is 8.8 ◦C, with the lowest temperature being −6.9 ◦C in January and the highest being 22.6 ◦C in July. The mean annual rainfall is 507 mm. The soil is classified as Calcaric Cambisol. Rainfall shows a highly seasonal variability with ca. 82% occurring from May to September (the growing season). The soil available N, P, and K were 20.9–71.3 mg kg−<sup>1</sup> , 1.6–2.8 mg kg−<sup>1</sup> , and 10.07–30.97 g kg−<sup>1</sup> , respectively, and soil pH was 8.4–8.8 [67]. The targeted grassland is dominated by xerophytic plants, e.g., *B. ischaemum*, *S. bungeana*, *L. davurica*, *Artemisia sacrorum*, and *Artemisia giraldii*.

#### *4.2. N and P addition*

A grassland community (20 × 30 m) was fenced to exclude grazing since May 2017. A randomized split-plot design with three N addition rates at the main plot level and three P addition rates at the subplot level was carried out. The main plot was 4 × 4 m, and N addition rates were N0 (0 kg N), N50 (50 kg N ha−<sup>1</sup> yr−<sup>1</sup> ), and N100 (100 kg N ha−<sup>1</sup> yr−<sup>1</sup> ). The N50 and N100 treatments were about 2 and 4 times the annual average N deposition rate in the loess hilly area [~21.76 kg (N) ha−<sup>1</sup> yr−<sup>1</sup> ] [68]. N was applied as calcium ammonium nitrate [5Ca(NO3)<sup>2</sup> NH4NO<sup>3</sup> 10H2O] (15.5% of N). Each main plot was divided into four subplots (2 × 2 m). P was applied as triple superphosphate [Ca(H2PO4)2·H2O] (45% of P), and the addition rates were set to 0, 1, and 2 times the local fertilization rate, corresponding to P0 (0 kg P2O5), P40 (40 kg P2O<sup>5</sup> ha−<sup>1</sup> yr−<sup>1</sup> ), and P80 (80 kg P2O<sup>5</sup> ha−<sup>1</sup> yr−<sup>1</sup> ) [44].

Totally, there were 9 treatments, including a control (N0P0), two N addition alone treatments (N50P0, N100P0), two P addition alone treatments (N0P40, N0P80), four N and P combined addition treatments (N50P40, N50P80, N100P40, N100P80), and three replicates per treatment. N and P additions were conducted once a year, on rainy days, from 2017–2019 (4 June 2017, 21 May 2018, and 13 June 2019).

#### *4.3. Ecophysiological Measurements*

#### 4.3.1. Diurnal Variations of Photosynthesis

The portable photosynthesis system (CIRAS-2, PP Systems, Amesbury, MA, USA) was used to measure the diurnal changes of photosynthesis of *B. ischaemum, S. bungeana*, and *L. davurica*, successively, and all measurements were conducted on three consecutive sunny days from 20–22 July 2019 (one species per day). The measurement was taken on one newly fully-expanded healthy leaf per species per treatment from 8:00–18:00 h with 2 h intervals. The measured parameters include net photosynthetic rate (*P*n, <sup>µ</sup>mol·m−<sup>2</sup> ·s −1 ), transpiration rate (*T*r, mmol·m−<sup>2</sup> ·s −1 ), intercellular CO<sup>2</sup> concentration (*C*<sup>i</sup> , <sup>µ</sup>mol·mol−<sup>1</sup> ), and environmental factors, including photosynthetically active radiation (PAR, <sup>µ</sup>mol·m−<sup>2</sup> ·s −1 ), air temperature (*T*a, ◦C), and relative humidity (RH, %). The photosynthetic rate at 10:00 h was taken as the maximum net photosynthetic rate (*P*Nmax, <sup>µ</sup>mol·m−<sup>2</sup> ·s −1 ). Instantaneous water use efficiency (WUE, µmol mmol−<sup>1</sup> ) was calculated as *P*n/*T*r. Stomatal limitation value (*L*s) was derived by 1−*C*i/*C*<sup>a</sup> [48].

#### 4.3.2. Leaf SPAD Value

Leaf SPAD value (a measure of leaf relative chlorophyll content) was measured on three newly fully-expanded healthy leaves per species per treatment using a chlorophyll meter (SPAD-502 model, Konica-Minolta, Osaka, Japan) on 20–22 July 2019.

#### 4.3.3. Leaf Functional Traits

The 10–20 newly-fully expanded healthy leaves were randomly sampled per species per treatment, stored in zipped plastic bags, and quickly taken back to the laboratory, in an insulated box with ice packs, for leaf functional traits measurements. Leaves were weighed with an analytical balance (d = 0.0001 g). The fresh leaves were scanned (Epson duplex scanner, Epson, Tokyo, Japan), and the leaf area was derived using ImageJ (National Institutes of Health, Bethesda, MD, USA). Then, leaves were oven-dried at 75 ◦C for 24 h and ground with a high-throughput tissue grinder (MM-400, Retsch, Haan, Germany). Specific leaf area (SLA, m<sup>2</sup> g –1) was calculated as leaf area divided by leaf dry mass. Leaf dry matter content (LDMC, g g–1) was calculated as leaf dry mass divided by fresh mass. After digestion with H2SO4-HClO4, the mass-based leaf N concentration (*N*mass) was obtained using a Kjeldahl N analyzer (FOSS-8400, Foss, Höganäs, Denmark). The massbased leaf P concentration *(P*mass) was determined by a molybdenum blue colorimetry (UV-2600 ultraviolet-visible spectrophotometer, Shimadzu, Kyoto, Japan). *Nmass/Pmass* ratio was then calculated.

#### *4.4. Statistical Analysis*

All statistical analyses were performed with SPSS 20.0. One-way analysis of variance (ANOVA) was used to compare the differences in leaf photosynthetic characteristics (WUE, *L*s, and *P*Nmax) and leaf functional traits (SPAD, *N*mass, *P*mass, *N*mass/*P*mass, SLA, and LDMC) of the three species under different N and P addition treatments. Tukey's HSD test was used for multiple comparisons. Two-way ANOVA was used to test the effects of N addition, P addition, and their interaction on *P*Nmax, SPAD value, *N*mass, *P*mass, SLA, and LDMC. Pearson correlation was used to explore the relationship between leaf photosynthetic characteristics (*P*Nmax, WUE) and leaf functional traits (SPAD value, *N*mass, *P*mass, *N*mass/ *P*mass, SLA, and LDMC). Multiple linear regression was used to explore the relationship between N addition, P addition, and their interaction, as well as *P*Nmax and WUE. Principal component analysis (PCA) was conducted on photosynthetic characteristics and leaf functional traits. Graphing was performed with Origin 2021 (Origin Lab Software, Chicago, IL, USA).

#### **5. Conclusions**

Our three-year field fertilization study suggested that N addition—alone or combined with P—improved the photosynthesis of the three grassland dominant species on the semiarid Loess Plateau of China. All three species shifted to a fast-growth strategy with increased *P*Nmax, SLA, and *N*mass, as well as reduced LDMC under N and/or P addition. Furthermore, species-specific shifts in leaf functional traits were observed among the three species following N and/or P addition, of which C<sup>4</sup> grass *B. ischaemum* increased SLA and *P*Nmax, and C<sup>3</sup> grass *S. bungeana* and subshrub *L. davurica* mainly increased leaf N and SPAD. P addition seems to only effectively impact the *P<sup>n</sup>* of *L. davurica*. Evident N and P synergetic effects on the photosynthetic performance in all three species were observed, and a combination of 50 kg ha−<sup>1</sup> yr−<sup>1</sup> N and 40 kg ha−<sup>1</sup> yr−<sup>1</sup> P addition could be considered optimal fertilization for improving grassland productivity locally.

**Author Contributions:** Conceptualization: B.X.; Methodology: Y.J., S.L. and Z.C.; data collection and curation: Y.J. and S.L.; data analysis: Y.J. and S.L.; field investigation: Y.J., S.L., C.J. and J.Z.; writing-review and editing: Y.J., F.N. and B.X. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was funded by National Key Research and Development Program of China (2016YFC0501703).

**Data Availability Statement:** The data presented in this study are available on reasonable request from the corresponding author.

**Conflicts of Interest:** The authors declare no conflict of interest.
