Nitrogen fertilization and CO₂ concentration synergistically affect the growth and protein content of Agropyron mongolicum

Introduction

Atmospheric carbon dioxide (CO₂) concentration has risen from 270 µmol mol⁻¹ before the industrial revolution to 414 µmol mol⁻¹ in 2020 (Domiciano et al., 2020), and the rate of increase is predicted to hasten during this century (IPCC, 2013). Elevated CO₂ concentrations (eCO₂) have a significant impact on plant growth, productivity, and quality in natural and agricultural systems (Burgess & Huang, 2014; Liu, Tian & Zhang, 2016). It is well established that plants can benefit from the augmented atmospheric CO₂ concentration through the “CO₂ fertilization effect”, particularly for C₃ species (Burgess & Huang, 2014). Since C₃ plants use the carboxylase enzyme RuBisCO (ribulose-1,5-bisphosphate carboxylase-oxygenase) to fix CO₂ from the air and obtain 3-carbon intermediate molecules as the first step in photosynthesis, lose a portion of their fixed CO₂ to oxidative photorespiration under present CO₂:O₂ ratios because RuBisCO is also an oxygenase (Ainsworth & Long, 2005; Reich et al., 2018). By contrast, in C₄ plants, a different enzyme (phosphoenolpyruvate carboxylase) with a high affinity for CO₂ and lacking oxygenase activity first incorporate CO₂ into a 4-carbon intermediate, which is then shuttled to specialized bundle sheath cells where CO₂ isreleased, and a high CO₂:O₂ ratio results in a lower rate of photorespiration. Thus, C₃ plants exhibit increased photosynthesis as increasing CO₂:O₂ ratios reduce rates of photorespiration and increase rates of carboxylation, while the photosynthetic rate of C₄ plants is hardly affected by eCO2 levels in the air (Reich et al., 2018). Meanwhile, the eCO₂ can decrease stomatal conductance (Gs) and transpiration rate (Tr) thereby increasing water use efficiency (Li, Wang & Liu, 2021), which can mitigate the negative climate effects accompanied by rising atmospheric CO₂ effects, such as global warming and precipitation pattern (Zheng et al., 2020). However, higher growth rates and substantial biomass accumulation dilute nutrients within their tissues. It has been reported that the nitrogen and protein concentrations in plant tissues exposed to eCO₂ generally declined due to constrain by N availability.

Previous studies have shown that the eCO₂ can decrease nitrogen (N) concentration in plant tissues, particularly under N-limited conditions. One reason for the lowered N concentration of plants grown under eCO₂ is the dilution of N by extra carbohydrate accumulation (Li, Wang & Liu, 2021). On the other hand, the uptake of N through transpiration-driven mass flow depends on the Tr and N ion concentration in the rhizosphere (Plhák, 2003). The eCO₂ could cause a decrease in N uptake rate per unit mass or length of root due to decreased Tr thereby decreasing the mass flow of ${\mathrm{NO}}_3^{-}$ from the soil to the root (Li, Wang & Liu, 2021; Mcdonald, Erickson & Kruger, 2002). For example, Bloom (2015) observed that lessened leaf protein content under eCO₂ is linked with limitations in ${\mathrm{NO}}_3^{-}$ assimilation caused by the reduction in transpiration. Also, eCO₂ induced N-deficiency and sink:source imbalance, especially in leaf scale, can aggravate another phenomenon that is commonly observed: photosynthetic acclimation to eCO₂ (Halpern et al., 2018). CO₂ acclimation is intrinsically related to a reduction of Rubisco, and a decrease in leaf gas exchange and carboxylation capacity (Erice et al., 2014), consequently resulting in decreasing carbon assimilation rates and limiting plant growth (Cohen et al., 2019; Zheng et al., 2019). Similarly, it has been previously described in meta-analyses that the reduction of leaf N content in plants responding to eCO₂ directly affects protein content (Loladze, 2014) and thus affects carbon fixation. The reduction of photosynthetic weakens, though generally does not eliminate, the expected stimulation of eCO₂ on growth (Jauregui et al., 2015).

Nitrogen (N) is the mineral element that plants require in the largest quantities (Fernando et al., 2017), and it is a key component of amino and nucleic acids (Cohen et al., 2019). Plants mainly acquire N from the soil as nitrate (${\mathrm{NO}}_3^{-}$ ) and ammonium (${\mathrm{NH}}_4^{+}$). Thus, fertilization with N is vital for plant growth and development. N-fertilization has been described to increase root growth, alter the shoot-to-root ratio, favor fine-root proliferation and modify root architecture of Arabidopsis plants (Jauregui et al., 2016). Such effects highlight the fact that roots play a central role in nutrient uptake and assimilation (BassiriRad, Gutschick & Lussenhop, 2001). In addition to the central role of the root, the increase in leaf N has also been suggested. As leaf N directly affects photosynthetic capacity and gas exchange, leaf N concentration is correlated to the Gs, Tr, photosynthetic rate (Pn), and growth (Lei et al., 2012). A large part of N in plant tissues is present in protein, so protein concentrations are often directly related to total N concentrations (Bahrami et al., 2017).

Although some measures and mechanisms to improve plant nitrogen content under eCO₂ have been reported on several occasions, such as protein extraction in the biorefinery (Solati et al., 2018), optimization the planting system (Manevski et al., 2017), improvement of photosynthesis and water use efficiency (Li, Wang & Liu, 2021; Manderscheid et al., 2018), increment of absorption and assimilation of ${\mathrm{NO}}_3^{-}$ (Bloom et al., 2014), as well as root formation and root elongation (Cohen et al., 2019), the combined effects of eCO₂ and different N-fertigation levels on plant growth and N nutrition remain largely elusive. Particularly rare are studies measuring the effects of eCO₂ and N on native grassland plant species in terms of productivity and N nutritional quality from root and leaf levels.

In the present study, we investigated the interactive effects of eCO₂ plus N-fertilization on the root, shoot biomass, root length (RL), leaf area (LA), specific leaf area (SLA), leaf nitrogen concentration (LNC), Pn, Gs, Tr, and physiological processes associated with N acquisition of the dominant species of Agropyron mongolicum, a perennial rhizomatous C₃ grass, in Ningxia desert steppe by growing them in soils collected from Ningxia desert steppe grassland using open-top chambers (OTCs). Our working hypothesis is that eCO₂ and N-fertilization would further enhance biomass accumulation while increasing plant N nutrition under eCO₂, where LNC, total root length (TRL), root N uptake capacity, root and leaf ${\mathrm{NO}}_3^{-}$ assimilation, as well as Gs and Tr, would play an important role in modulating the effects of the fertigation with N on plant growth and N nutritional quality under eCO₂.

Materials and Methods

Results

Biomass, root, and leaf morphology traits

Biomass of both shoot and root in A. mongolicum exhibited consistently faster growth in all N treatments compared to N0 under both CO₂ conditions (Figs. 1A and 1B). Compared with aCO₂, eCO₂ exerted greatly growth stimulation under different N treatments, with a 30.8% increase only in the shoot biomass under low N, whereas 39.9% and 107.6% stimulation under high N in root and shoot biomass, respectively. The different responses of the shoot and root biomass in A. mongolicum to enriched CO₂ andN led to a decline in the root/shoot ratio. The root/shoot ratios of A. mongolicum were 43.4%, 43%, and 42.2% under aCO₂ at control, low, and high N treatments, remaining relatively constant changes; whereas under eCO₂, the ratios were increased to 47.9%, 43.9%, and 38.1%, respectively. The results of two-way ANOVA indicated that CO₂ concentration and N-fertilization level had significant effects on the shoot and root biomass, but their interaction had no significant effects. However, changes in root/shoot ratio were depending on N and interactions between CO₂ concentration and N fertilization, while CO₂ concentration had no impact on the root/shoot ratio.

Leaf nitrogen concentration (LNC) and leaf area (LA) of A. mongolicum were significantly increased under both CO₂ conditions at low and high N treatments (Figs. 1D and 1E), while the same treatments caused a significant reduction in SLA of A. mongolicum (Fig. 1F). High N supplement increased LNC and LA in A. mongolicum by 49.3% and 69% at aCO₂, and by 53.9% and 65.7% at eCO₂, while decreased 11.1% and 28.2% in the SLA under both CO₂ conditions, respectively, compared to control treatment. Moreover, both CO₂ concentration and N addition level had significant effects on the LA and SLA, but LNC was only affected by N-fertigation, the interaction between CO₂ concentration and N levels had no significant effect on any one of these variables.

N-fertilization stimulated the root morphology of A. mongolicum under both aCO₂ and eCO₂, for TRL, a 150.1% and 100.4% increase at high N treatment (Fig. 1G), whereas153.9% and 144.7% stimulation in RSA with high N treatment at aCO₂ and eCO₂ (Fig. 1H), respectively. There was no significant difference in root N uptake capacity under aCO₂, while it’s significantly increased with increasing N supplement under eCO₂, with a 61.6% increase at high N level (Fig. 1I). Furthermore, both CO₂ concentration and N-fertilization level had significant effects on the TRL and RSA, their interaction had only effect on RSA, while root N uptake capacity was only affected by N-fertigation.

Leaf gas exchange

Net photosynthetic rate (Pn) and transpiration rate (Tr) were significantly affected by CO₂, N-fertigation, and the interaction between CO₂ concentration and N-fertilization (Figs. 2A and 2C), while stomatal conductance (Gs) was significantly affected by N-fertilization and their interaction (Fig. 2B). The Pn of A. mongolicum increased by 80.8% and 26.9%, respectively, at high N treatment compared to that at the control treatment under both aCO₂ and eCO₂. N-fertigation promoted the increase of the Gs and Tr under ambient and elevated CO₂, although not statistically significant in eCO₂ condition. High N-fertigation increased the Gs and Tr of A. mongolicum by 107.4% and 109% at aCO₂, respectively, compared to control treatments.

Total N, amino acid, protein, NR, and GS activity in root and leaf

The content of total nitrogen (TN), free amino acid, and protein in root and shoot was significantly affected by CO₂ concentration and N-fertilization (Figs. 3A, 3B and 3C), the eCO₂ increased TN of root and shoot by 50.6% and 58.3%, respectively, compared to aCO₂. The TN of root and shoot grown under high N at aCO₂ conditions was increased by 205.5% and 213.9%, respectively, but by 134.4% and 223.1% at eCO₂, compared to that under control treatment. However, the interaction between N and CO₂ did not have significant influences on these variables in both root and shoot, except for protein content in the root. When comparing the effect of N-fertigation on the free amino acid content, the increase observed in the root and leaf were 62.9% and 16.1% at aCO₂ and 66.5% and 16.1% at eCO₂ under high N treatment, respectively, compared to control treatments. The content of protein in leaf and root progressively increased from control to high N treatment, with high N treatment increasing protein content in leaf by 20.3% and 31.3% under both a CO₂ and eCO₂, respectively, compared to control treatment, and this increase was higher for eCO₂ than aCO₂ plants.

The ratio of nitrate to total nitrogen (${\mathrm{NO}}_3^{-}$/TN) in leaf decreased by 5.5% and 21.9% at high N under eCO₂ and aCO₂, respectively, compared to control treatment, while decreased 38.5% and 22.6% in root under both CO₂ conditions. However, this decrease did not differ among N treatments under aCO₂ (Fig. 3D). The NR in leaf and root was significantly increased with increasing N addition under both CO₂ conditions. Compared to control treatment, high N treatment increased NR in leaf by 162.6% and 69.4% and by 67.8% and 96.7% in root under both a CO₂ andeCO₂ (Fig. 3E), respectively. There was an increase in GS activity in leaf and root for all the N treatments when exposure to both CO₂ conditions, GS activity in leaf and root treated with high N treatment was 67.8% and 71.9% at eCO₂, respectively, higher than that of control treatment and same N treatments at aCO₂ conditions (Fig. 3F). In addition, except for ${\mathrm{NO}}_3^{-}$/TN in root only affected by N-fertilization, the ${\mathrm{NO}}_3^{-}$/TN, GS, and NR activity, across all leaf and root, was significantly affected by CO₂ and N-fertigation, respectively, while the interaction between N and CO₂ only influenced the ${\mathrm{NO}}_3^{-}$/TN of leaf and the NR in leaf and root.

Multivariate analysis of traits for growth and N nutrient of A. mongolicum

Constrained redundancy analysis (RDA) performed for A. mongolicum indicated that the first two axes explained 84.5% of the total variance (Fig. 4A). Root and shoot biomass showed positive correlations with LNC, RNU, Pn, RTN, STN, SPC, and RPC were positively related to LNC, Gs, Tr, RNU, L-GS, R-GS R-NR, L-NR; whereas, except for the above, RPC and RTN showed negative correlations with L- ${\mathrm{NO}}_3^{-}$/TN and R- ${\mathrm{NO}}_3^{-}$/TN, respectively (Table S1). These findings indicated that the growth and N nutrient content of A. mongolicum were principally influenced by root and leaf morphophysiological as well as biochemical characteristics under eCO₂ and N-fertilization.

To further reveal the correlations between the various traits and both growth and N nutrient, correlation networks were constructed (Figs. 4B and 4C). The results also showed that L-GS, Gs, Pn, and RNU was strongly and positively correlated with the shoot and root biomass (r > 0.5). Furthermore, the STN was strongly and positively correlated with the LNC, RNU, Tr, Gs, R-NR, L-NR, R-GS, and GS (r > 0.5); the RTN was strongly and positively correlated with the RNU, Tr, Gs, R-NR, L-NR, R-GS, L-GS, and GS (r > 0.5); while they were mediumly and negatively correlated with L- ${\mathrm{NO}}_3^{-}$/TN and R- ${\mathrm{NO}}_3^{-}$/TN, respectively (0.3 ≤ r < 0.5). Similar results have also been found in total protein content, the SPC and RPC were a strongly and mediumly positive correlation with the LNC, RNU, R-NR, R-NR, R-GS, and L-GS (r ≥ 0.3), and they were a small negative correlation with SLA and R- ${\mathrm{NO}}_3^{-}$/TN ( r < 0.3), respectively.

Discussion

This study clearly showed that the increase in atmospheric CO₂ played a major role in A. mongolicum growth and N nutrition and that this role is determined by the available N. This interaction demonstrated that the responses of plants to the eCO₂ and N-fertilization are dependent on the coordination between root and leaf development and are associated with the metabolic implications of the available N. Our study showed that although both CO₂ conditions increased root and shoot biomass, eCO₂ combined with the increased N supplement accelerated the effect of the eCO₂ on the growth stimulation of plants, demonstrating that the promotion of A. mongolicum growth under the short-term enrichment of air CO₂ is dependent on the available N (Figs. 1A and 1B). A previous study has shown that plant maximum growth response to eCO₂ could be improved by the effects of eCO₂ on the leaf carboxylation rate per unit leaf area or relative growth rate when plants received adequate N for growth (Leakey et al., 2009; Liu et al., 2012). Our growth stimulation can be partly explained by the increment in leaf photosynthesis that occurs when A. mongolicum is grown under eCO₂ plus N-fertilization. The SLA expressed as the leaf area per unit of dry weight is one of the most widely used traits for describing leaf characteristics and their relationship with photosynthesis (Burgess & Huang, 2014). In our study, the combination of higher leaf N content and a lower SLA indicated that A. mongolicum developed under eCO₂ plus N-fertilization conditions had a smaller leaf area per unit biomass or the leaves become thicker, which was consistent with some studies (Burgess & Huang, 2014; Lei et al., 2012; Liu et al., 2012). It was shown that tree species grown in elevated CO₂ combined with increased N-fertilization had thicker leaves and a greater leaf weight per unit leaf area (Liu et al., 2012). It was suggested that the thicker leaves with increased mesophyll and cell N content of leaves may be a reason for the observed increase in photosynthetic rates (Jauregui et al., 2016). Our study confirmed the conclusion that leaf area per unit biomass decreased and the photosynthetic rate increased upon exposure to 800 µmol mol⁻¹ CO₂ (Figs. 1F and 2A). Furthermore, leaf thickness has also been shown to have a close relationship with the transpiration rate wherein thicker leaves have greater transpiration efficiency, which was to be beneficial to the nutritional uptake of plants (Giuliani et al., 2013), increased N content in the leaves in eCO₂ could also alleviate photosynthetic acclimation to CO₂ enrichment, enhancing photosynthetic rates of leaves (Vicente et al., 2015).

The increased shoot biomass in A. mongolicum was mainly due to enhanced leaf photosynthesis, as discussed above. Likewise, roots, such as root morphology and architecture, also played an important role in plant growth responses to eCO₂ (Burgess & Huang, 2014). A review described the effects of eCO₂ on plant root systems and reported that both the root number and root length are significantly increased due to eCO₂ and other interacting factors, such as warming, precipitation, and nutrient availability (Leakey et al., 2009). In a comprehensive review, it was also reported that other structural aspects of root growth tend to increase when plants are maintained at eCO₂ levels, including the volume, branching, and relative growth rate, whereas reports of changes to the root N uptake capacity due to eCO₂ level are lacking (Rogers, Runion & Krupa, 1994). As expected, root length, root surface area, and N uptake capacity of A. mongolicum grown under at eCO₂ various N conditions were significantly increased compared to that at the same N treatment under aCO₂ (Figs. 1G and 1H), and the degree of increase relative to ambient controls was highly dependent upon the N availability. The stimulation of root growth can have a substantial effect on plant growth due to greater soil volume exploration in deeper soil strata. Most interestingly, with the increase in N treatments, the response of the root/shoot ratio of A. mongolicum to changes in CO₂ concentrations was contrary to the finding of studies with increases in R/S previously reported (Cohen et al., 2019). However, this is less surprising when we considered the available resources since increased root growth requires both C and N inputs. It has been reported that plants with adaptation to low N treatment often allocate relatively more biomass to roots and enhanced root nutrients uptake capacity, consequently, eCO₂ enhances root growth more than shoot growth(Lei et al., 2012; Liu, Tian & Zhang, 2016). With the high N treatment, however, the increase in available C due to increased photosynthesis, coupled with a non-limiting N supply, made it possible to increase the shoot growth substantially (Vicente et al., 2015). Root proliferation through increased length or surface area serves as a critical adjustment function for water or nutrients uptake and adaptation mechanisms to an increased rate of carbon and nitrogen cycling have been implicated in enhancing plant productivity or nutritional quality in an agro-ecosystem (BassiriRad, Gutschick & Lussenhop, 2001; Leakey et al., 2009; Liu et al., 2012).

It is well known that eCO₂ induces stomatal closure can decline Gs and Tr of plants while decreasing the transpiration-driven mass flow of N, consequently decreasing N uptake by roots (Jayawardena et al., 2021; Li, Wang & Liu, 2021; McGrath & Lobell, 2013). Consistent with this, the results from the present study showed that eCO₂ significantly decreased Gs and Tr compared with aCO₂ (Figs. 2B and 2C), however, high N supply increased Gs and Tr to approximately 1.5 and 1.6 times that of control N treatments, respectively, regardless of the CO₂ treatments. Accumulated evidence has revealed that increasing the nutrient supply from low to high can increase plant N uptake by increasing Gs as well as Tr of plants (De Oliveira et al., 2012; Lotfiomran, Kohl & Fromm, 2016), consistent with the results from the present study. Therefore, we suggest that the increase of both stomatal Gs and Tr by adding N under eCO₂ could weaken the negative effect of eCO₂ in decreasing the transpiration-driven mass flow of N.

The protein decrease under eCO₂ was not only related to the external growth development limitations to N uptake but also internal signals arising from the progress imposed by physiological metabolism, most prominently inhibition of ${\mathrm{NO}}_3^{-}$ assimilation (Bloom et al., 2014). The ${\mathrm{NO}}_3^{-}$/total N ratio was used as a surrogate for plant unassimilated ${\mathrm{NO}}_3^{-}$ and negatively correlated with total protein in plant tissues (Bahrami et al., 2017; Jayawardena et al., 2021). The present study showed that increasing N-fertilization did not influence the leaf ${\mathrm{NO}}_3^{-}$ to total N ratio at aCO₂, but it decreased the leaf ${\mathrm{NO}}_3^{-}$ to total N ratio at eCO₂ and root ${\mathrm{NO}}_3^{-}$ to total N ratio under both CO₂ conditions (Fig. 3D), and shoot and root protein content negatively correlated with L- ${\mathrm{NO}}_3^{-}$/TN and R- ${\mathrm{NO}}_3^{-}$/TN, respectively (Figs. 4A and 4B), indicating N-fertilization could alleviate the effects of eCO₂ on inhibition of ${\mathrm{NO}}_3^{-}$ photo-assimilation of A. mongolicum. The proportion of unassimilated ${\mathrm{NO}}_3^{-}$ was declined, either by assimilation of ${\mathrm{NO}}_3^{-}$ or by an increase in total N, or both (Bahrami et al., 2017). With increased N supply, the total N content and the free amino acids in leaves and roots of A. mongolicum significantly increased when plants were exposed to two CO₂ conditions (Figs. 3A and 3B), suggesting the above both occurred. Supporting the possibility of enhancement of ${\mathrm{NO}}_3^{-}$ assimilation, we saw an increase in the concentrations of N-assimilatory proteins, such as NR and GS activities (Figs. 3E and 3F), at eCO₂ and N-fertilization, and positively correlated with LPC and RPC (Fig. 4B), which is in agreement with previous studies (Bahrami et al., 2017; Jauregui et al., 2016; Jayawardena et al., 2017). Croy & Hageman (1970), who used increased NR activity as a proxy for a decline of unassimilated ${\mathrm{NO}}_3^{-}$, explained a positive correlation between leaf NR activity and grain protein in wheat. Notably, the NR and GS activity in leaves and roots were able to maintain relatively similar levels among all same treatment combinations; therefore, we inferred that the proportion of ${\mathrm{NO}}_3^{-}$ in total N in roots which appeared to be more highly than in leaves might be mainly because of the large increase in total N content in leaves. Furthermore, as previous studies discussed, the positive effect of eCO₂ on plants growth during different N sources was significantly correlated with ${\mathrm{NO}}_3^{-}$ uptake and assimilation (Domiciano et al., 2020; Jayawardena et al., 2017). Taken together, our results suggest that N-fertilization could alleviate inhibition of ${\mathrm{NO}}_3^{-}$ assimilation under eCO₂ by increasing concentrations of N-assimilatory proteins and enhancing ${\mathrm{NO}}_3^{-}$ assimilation, consequently, improving productivity and quality of A. mongolicum.

Conclusions

N-fertigation causes photosynthesis improvement via increased leaf N content and reduced specific leaf area, resulting in enhanced stimulation for growth by eCO₂, but this stimulation does not affect the N nutrition of A. mongolicum under eCO₂. Under sufficient N supply, the reduced proportion of ${\mathrm{NO}}_3^{-}$ in total leaf and root N, increased leaf N content, root length, and Tr are not only associated with N concentrations in vegetative plant parts, but also with the total protein content of A. mongolicum grown at eCO₂. Therefore, with the application of effective management measures of N-fertilization, grass productivity and quality may enhance under the CO₂ levels anticipated during the end of this century.

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