Background

It is widely evident that global atmospheric carbon dioxide (CO2) concentration has dramatically increased since the nineteenth century industrial revolution, elevating by about 1.6 ppm/yr. during the past five decades [1, 2]. According to the most recent report released by the Inter-Governmental Panel on Climate Change (IPCC, 2013), global atmospheric CO2 levels have increased from the pre-industrial level of 280 ppm to the present level of nearly 410 ppm and the growth rate of CO2 concentration is projected to be accelerated with an unprecedented pace of ∼1.0 ppm/yr. [2,3,4]. Moreover, the global atmospheric CO2 concentration may even reach 1000 ppm by the end of this century and nearly 2000 ppm by the end of the next century if no effective control measures are implemented [4]. This elevated global atmospheric CO2 concentration may not only cause climate warming, but also cause profound impacts on the net primary productivity of agricultural and natural ecosystems [5,6,7,8,9].

It is well known that CO2 is not only one of the most important greenhouse gases, but also a critical reactant for the biochemical processes of plant photosynthesis, and thus future elevated CO2 concentrations may affect plant growth by altering metabolic rates [10,11,12,13]. Many studies have reported that most plants may benefit from enriched atmospheric CO2 concentrations through the “CO2 fertilization effect”. Plant growth can be boosted by absorbing more CO2 molecules for photosynthesis under elevated CO2 concentrations [10, 14,15,16,17]. For example, Wand [18] reviewed the responses of wild grasses to elevated atmospheric CO2 concentrations and found that elevated CO2 increased the total biomass of C3 grass species by about 50%. However, other studies have shown that the CO2 fertilization effect on plant growth might decline or vanish beyond certain CO2 concentrations [7, 19, 20], and even CO2 enrichment induced adverse effects on some plants when the ambient CO2 level was above 1000 ppm [21]. In addition, many previous studies also found that the CO2 fertilization effect on plants had a large variation among different species. For example, Wang [18] reported a substantial increase of the biomass of young birch tree by 59% when CO2 concentration was doubled from about 350 ppm to 700 ppm. By contrast, Körner et al. [22] showed that the growth and biomass of five tree species in a mature deciduous forest were barely affected by increasing CO2 concentration to 530 ppm based on a four-year FACE experiment. These results indicate that different plant species may have different optimal CO2 concentrations, and that plants with higher optimal CO2 concentrations are likely to benefit the most from the CO2 fertilization effect, and at the same time, suffer less negative impacts from future climate change, mainly due to higher nitrogen and water use efficiency [23, 24].

The CO2 fertilization effect on plant growth was fundamentally mediated by leaf photosynthesis [19, 25, 26], which is highly correlated with plant carbon balance [27] and biochemical composition [28, 29]. Previous studies have demonstrated that elevated CO2 could dramatically affect net photosynthetic rates through various processes including up-regulation or down-regulation when the growth CO2 below or above the optimal CO2 for plants. Elevated CO2 levels generally stimulate net photosynthetic rate through directly enhancing carboxylation rates [13, 30] while competitively reducing photorespiration and dark respiration [19, 22, 31,32,33]. Nevertheless, the decline of net photosynthetic rate under high CO2 levels may be related to changes in leaf biochemical composition associated with reductions in the amount and/or activity of Rubisco [22, 26], and increases in total non-structural carbohydrates [7, 34]. Moreover, the down-regulation of net photosynthetic rate is also associated with the availability of nutrients such as nitrogen (N), which exerts an important control over the response of plants and ecosystems in rising atmospheric CO2 conditions [28, 35,36,37]. Previous studies showed that down-regulation of photosynthesis occurred in plants grown in elevated CO2 and limited N indicated decreased leaf N concentration [38, 39] High N availability could alleviate the down-regulation of photosynthesis in plants under elevated CO2 environments [19, 26, 29].

Grasslands are an important part of terrestrial ecosystems, and account for about 20% of the earth’s land area [6, 40]. Perennial grasses are the dominant species in temperate grasslands and pastures [40], and are utilized as fine turf grass, which serves many important environmental functions including erosion control, surface water detoxification and control of allergens and diseases [41, 42]. A majority of the research investigating plant response to elevated CO2 have been focused on crops [43,44,45] or trees [26, 29, 34, 46,47,48] and few studies have examined the effects of elevated CO2 on perennial grasses [17, 19, 40]. In addition, most previous studies regarding the CO2 fertilization effect have focused primarily on “doubling-CO2 experiments” with twofold higher CO2 concentration of about 700 or 800 ppm than the current global CO2 concentration [40, 42, 45, 48]. Nevertheless, the CO2 fertilization effect may sustain up to about 1000 ppm for leaf photosynthesis [46, 49] and 1800 ppm for grain yield of crops [50]. For example, Xu [23] examined the optimal atmospheric CO2 concentration of the CO2 fertilization effect on the growth of winter wheat and found that the optimal atmospheric CO2 concentration was 894 and 968 ppm for total biomass and leaf photosynthesis. So far, few experimental studies have been conducted to examine the optimal CO2 concentration for maximizing the CO2 fertilization effect on perennial grasses, which are the most important grass species in both natural grasslands and managed turf grass. Moreover, most of the modeling projections are based on strong CO2 fertilization according to the conclusions from earlier “doubling-CO2 experiments” [29, 34]. However, it should be noted that in the future, continuously rising atmospheric CO2 concentrations may substantially lower the CO2 fertilization effect when the atmospheric CO2 concentration rises beyond the optimal CO2 level [23]. As a result, many current climate change models based on earlier results of the doubling-CO2 experiments may overestimate the CO2 fertilization effect and underestimate the potential risks that climate change poses on global grasslands when the atmospheric CO2 concentration goes beyond the optimal CO2 level. Therefore, identifying optimal CO2 concentrations and understanding the mechanisms that determine these optima are not only critical to accurately estimating the impacts of climate change on global grassland production, but also have important significance for policy implementations under future climate change scenarios. Therefore, this study was conducted based on the following objectives: (1) investigate the effects of elevated CO2 concentrations on the growth of three perennial grass species, (2) examine the optimal CO2 concentration for maximizing the CO2 fertilization effect of these grasses, and (3) explore potential mechanisms that determine the optimal CO2 concentrations for the growth of perennial grasses.

Methods

Plant materials and growing conditions

Three grass species, tall fescue (Festuca arundinacea Schreb.), perennial ryegrass (Lolium perenne L.), and Kentucky bluegrass (Poa pratensis L.), were collected using a golf-hole cutter (10 cm diameter × 20 cm long) to ensure the same aboveground and belowground biomass of each species from field plots in the research farm at Rutgers University (Adelphia, NJ, USA). These grasses were irrigated with groundwater once a week in the field research farm to maintain a 10-cm soil surface moisture of about 40% (% volume) during the growing season. Then the collected plants were transplanted into pots (10 cm diameter × 40 cm long) filled with fritted clay and maintained in a greenhouse with an average temperature of 21/16 °C (day/night) and about 800 μmol photon m− 2 s− 1 Photosynthetic Active Radiation (PAR) in natural sun light, and 65% relative humidity for 70 d (May–June 2012) to establish canopy and root system. During the establishment period, grasses were irrigated daily to water-holding capacity and fertilized twice per week with half-strength Hoagland’s solution [51]. We trimmed grasses once a week to maintain a canopy height of 5 cm during the canopy development and root establishment period. Then the plants were trimmed to a 2-cm canopy height and moved to growth chambers (Environmental Growth Chamber) with temperatures set at 21/18 °C (day/night), 60–70% Relative Humidity (RH), light level at grass canopy of 1000 μmol m− 2 s− 1 PAR, and a 12-h photoperiod for 2 weeks prior to the CO2 treatment. During the eight weeks of the CO2 treatment, these grasses were maintained under the same environmental factors as before the start of CO2 treatment, such as chamber temperature of 21/18 °C (day/night), relative humidity of 60–70%, light level at the grass canopy of 1000 μmol m− 2 s− 1 PAR, and 12-h photoperiod (6:00–18:00). In addition, the grasses were also well-watered with daily irrigation and fertilized with half-strength Hoagland’s solution twice a week.

Treatments and experimental design

We exposed grasses to five CO2 treatments: ambient concentration (400 ± 10 ppm) or elevated concentrations (600, 800, 1000, and 1200 ± 10 ppm). In order to minimize confounding effects of environmental variation between different chambers, we randomly changed the CO2 concentration of each growth chamber every three days, and then relocated the CO2 treated grasses to the growth chambers with corresponding CO2 concentrations. The experiment was arranged in a randomized complete block design with four replicates (pots) per treatment. The ambient and elevated CO2 concentrations within the chambers were maintained through an automatic CO2 control system connected to a CO2 source-tank containing 100% research-grade CO2 (Airgas, Inc.). The CO2 concentrations inside the chambers were continuously monitored through an infrared gas analyzer (LI-820; LICOR, Inc., Lincoln, NB, USA) connected to a computer logger maintaining the CO2 concentration within 10 ppm of the ambient and elevated target levels.

Plant biomass measurements

We trimmed the plants to a 2-cm canopy height again at 14, 28, 42, and 56 days after the CO2 treatments. The trimmed leaves were collected and oven dried at 80 °C for 7 days, and the dry weights were subsequently measured. The dry weights of leaves collected at 14, 28, 42, and 56 days of CO2 treatment were put together for calculating shoot biomass during the CO2 treatment period. At the end of the treatment period (56 days), all plant samples were destructively removed for an analysis of root biomass accumulation. The roots were severed from the shoots at the soil line and washed to make free of fritted clay medium. All of the washed roots were then oven dried at 80 °C for 3 days, and the dry weights were subsequently measured.

Leaf gas exchange measurements

Leaf gas exchange measurements were performed at the end of the CO2 treatment period (56 days). Five fully expanded leaves were randomly selected and arranged in a 2 × 3 cm2 cuvette chamber attached to a portable photosynthetic system (LI-6400; LICOR, Inc.). Before each measurement, leaves were equilibrated in the cuvette at saturating PPFD (1000 μmol photon m− 2 s− 1), the growth CO2 level, the target temperature and Vapor Pressure Deficit (VPD). CO2 concentrations in the cuvette were controlled using an injector system (LI-6400, LI-COR Inc.), which utilizes a CO2 mixer and compressed CO2 cartridges sealed with plasticene to prevent leakage. Then, the photosynthesis vs intercellular CO2 (An-Ci) curves were measured at cuvette chamber CO2 of 50, 100, 150, 200, 300, 400, 600, 800, 1000, 1200, and 1400 ppm. Data from An-Ci curves were used to compare treatment effects on the light-saturated net photosynthetic rates at ambient or elevated CO2 (An), the maximum carboxylation rate of Rubisco (Vcmax), and the maximum capacity of electron transport mediated ribulose bisphosphate (RuBP) regeneration (Jmax). An estimation method was used to obtain Vcmax and Jmax for each observed An-Ci curve [52]. Meanwhile, stomatal conductance (gs), and transpiration rate (Tr) were also determined with the portable photosynthesis system (LI-6400; LICOR, Inc.). Water Use Efficiency (WUE) was determined by the values of the net photosynthetic rate (An) and transpiration rate (Tr) according to the formula WUE = An / Tr.

Biochemical analysis

After the CO2 treatment period (56 days), the leaves and roots for analyzing Total Non-structural Carbohydrates (TNC) were sampled at midday, immediately frozen in liquid nitrogen and stored at − 80 °C until freeze-drying. Freeze-dried tissues were then ground to fine powder with a ball mill (MM2, Fa. Retsch, Haan, Germany), applied desiccant and stored at 20 °C. Total carbon (C) and nitrogen (N) contents in leaves and roots were determined using an elemental analyzer (Vario Max CN, Elemnetar Corp., Germany). Glucose, fructose, sucrose and starch concentrations were determined spectrophometrically (UV-1750, Shimadzu Corp., Tokyo, Japan), using a glucose kit (GAHK-20, Sigma, St Louis, MO, USA). Phospho-glucose isomerase (P5381–1 KU, Sigma) and invertase (I-4504, Sigma) were used to convert fructose to glucose and sucrose to glucose respectively. Biochemical analyses were repeated five times and expressed on a percentage dry matter basis for each.

Data analysis

The raw data from the leaf photosynthesis measurements was cleaned and processed in Excel spreadsheets where the non-linear An-Ci curve fitting was performed as in Sharkey et al. (2007) [52]. The net assimilation rate (An) versus intercellular CO2 concentration (An-Ci curve), were fitted to estimate the maximum carboxylation rate (Vcmax), maximum electron transport rate (Jmax) based on the measurements of An-Ci curves. In addition, linear and non-linear (quadratic equations) regressions were employed to examine relationships between CO2 concentration and other variables.

Results

Elevated CO2 effects on plant biomass

We found very strong CO2 fertilization effects on the aboveground and total biomass of the three species. The optimal CO2 levels for the aboveground biomass were 945, 915, and 1151 ppm, and for the total biomass were 915, 1178, and 1386 ppm for tall fescue, perennial ryegrass, and Kentucky bluegrass, respectively (Fig. 1). However, an optimal CO2 of 895 ppm for the belowground was found only for the tall fescue, while no obviously optimal CO2 of the belowground biomass for the other two species was detected. Beyond the optimum, further elevating the ambient CO2 concentration significantly reduced the growth of perennial grasses, indicating the adverse impacts of high CO2 concentration on the grass species. Quadratic models can be used to adequately quantify the CO2 fertilization effect on the biomass of the three grasses (Fig. 1).

Fig. 1
figure 1

Effects of elevated CO2 concentrations on above ground biomass (a), below ground biomass (b), and total biomass (c) of the three grass species. Values given are mean ± standard deviation for n = 4 pots

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Elevated CO2 effects on leaf gas exchange

As with plant growth, the CO2 fertilization effect was also evident in the leaf net photosynthetic rate (An) of Kentucky bluegrass, stimulating An by 75% when the CO2 increased from 400 ppm to 1000 ppm. The CO2 stimulation effect on An reached a maximum at 959 ppm, at which point further increase in CO2 resulted in a decline of An (Fig. 2). However, the response of An to elevated CO2 also varied with grass species. The leaf net photosynthetic rates of the other two species (tall fescue and perennial ryegrass) consistently increased with increasing CO2, which can also be described by quadratic relationships with optimal CO2 beyond the maximum CO2 treatment of this study. In contrast to An, the stomatal conductance (gs) and transpiration rates (Tr) of the three grasses decreased non-linearly with the increase of CO2 and the relationships of CO2-gs and CO2-Tr also typically followed quadratic equations with maximum gs and Tr occurring around 400 ppm, which was much lower than the optimal CO2 for plant growth and leaf photosynthesis.

Fig. 2
figure 2

Effects of elevated CO2 concentrations on (a) leaf net photosynthesis rate (An), (b) water use efficiency (WUE), (c) stomatal conductance (gs), and (d) transpiration rate (Tr) of the three grass species. Values given are mean ± standard deviation for n = 4 pots

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As a result, the WUE of tall fescue and Kentucky bluegrass also featured bell-shaped curves in relation to CO2 concentration, with the maximum CO2 fertilization effect occurring at approximately 1062 ppm and 910 ppm, respectively. However, the maximum WUE of perennial ryegrass was beyond the highest CO2 concentration treatment of 1200 ppm. Thus, we quantified the relationship between CO2 and WUE of perennial ryegrass through quadratic models and found that the optimal CO2 for WUE would occur at about 2700 ppm, which was much higher than those of the other two species (Fig. 2).

The maximum carboxylation rate (Vcmax) of the three grasses demonstrated bell-shaped curves in relation to CO2 concentration, peaking at 906 ppm, 863 ppm, and 743 ppm for tall fescue, perennial ryegrass, and Kentucky bluegrass, respectively (Fig. 3a). Similar to the Vcmax, the maximum electron transport rate (Jmax) in response to increasing CO2 concentrations also shared bell-shaped curves for all three grasses. The optimal CO2 concentration of Jmax was 877 ppm, 941 ppm, and 665 ppm for tall fescue, perennial ryegrass, and Kentucky bluegrass, respectively (Fig. 3b).

Fig. 3
figure 3

Effects of elevated CO2 concentrations on (a) the maximum carboxylation rate (Vcmax) and (b) the maximum capacity of electron transport RuBP regeneration (Jmax) of the three grasses. Values given are mean ± standard deviation for n = 4 pots

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Elevated CO2 effects on leaf dark respiration and non-structural carbohydrates

Our results showed that leaf dark respiration (Rd) of the three species substantially declined with increasing CO2 (Fig. 4). The relationships between Rd and CO2 of the three species were quantified through quadratic models with R2 values of 0.99, 0.99 and 0.94 for tall fescue, perennial ryegrass and Kentucky bluegrass respectively (Fig. 4a). Similar to the Rd, the leaf total non-structural carbohydrate (TNC) of the three grasses also quadratically decreased with elevated CO2 (Fig. 4b). Meanwhile, we estimated the relationships between Rd and TNC (Fig. 5) and found that Rd was increased linearly by the enhancement of TNC, with R2 values such as 0.73, 0.78 and 0.95 for the tall fescue, perennial ryegrass, and Kentucky bluegrass, respectively.

Fig. 4
figure 4

Effects of elevated CO2 concentrations on (a) the leaf dark respiration rates (Rd) and (b) total non-structural carbohydrates (TNC) of the three grass species

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Fig. 5
figure 5

Effects of elevated CO2 concentrations on the linear relationship between TNC and Rd for tall fescue (a), perennial ryegrass (b), and Kentucky bluegrass (c)

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Elevated CO2 effects on tissue carbon (C) and nitrogen (N) contents and the relationships between leaf N and V cmax or leaf N and J max

We found optimal CO2 concentrations in both the leaf and root of tall fescue and perennial ryegrass. The relationships between leaf carbon and CO2 featured bell-shaped curves with maximum values occurring at approximately 1388 and 1600 ppm for tall fescue and perennial ryegrass with R2 values 0.96 and 0.99 respectively (Fig. 6a). Interestingly, root carbon in response to elevated CO2 was also characterized by similar curves with R2 values 0.71 and 0.78 and optimal CO2 levels of 1011 and 1200 ppm for tall fescue and perennial ryegrass, respectively. However, we obtained very weak relationships between CO2 and tissue carbon with R2 values 0.23 for leaf and 0.19 for root of Kentucky bluegrass (Fig. 6a). In contrast to tissue carbon, both the leaf and root nitrogen of the tall fescue and Kentucky bluegrass quadratically decreased with elevated CO2 (Fig. 6b). By using the quadratic functions, we analyzed the relationships of leaf and root nitrogen with CO2 and found the R2 values to be 0.79 and 0.71, and 0.31 and 0.44 for the tall fescue and Kentucky bluegrass respectively (Fig. 6d). Our results also showed that elevated CO2 barely affected the tissue nitrogen of perennial ryegrass, evidenced by the weak quadratic relationships between CO2 and nitrogen with R2 values 0.03 and 0.04 for leaf and root respectively (Fig. 6c-d).

Fig. 6
figure 6

Effects of elevated CO2 concentrations on the carbon content of root (a) and leaf (b) as well as the nitrogen content of root (c) and leaf (d) of the three grass species. Values given are mean ± standard deviation for n = 4 pots

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We also evaluated the relationships between leaf N and Vcmax as well as leaf N and Jmax of the three grass species (Fig. 7). Our results showed that the Vcmax values were linearly enhanced with the increases of leaf N for tall fescue (R2 = 0.70), perennial ryegrass (R2 = 0.70), and Kentucky bluegrass (R2 = 0.65, Fig. 7a-c). Similarly, we also found linearly positive relationships between leaf N and Jmax with R2 values of 0.57, 0.55, and 0.62 for tall fescue, perennial ryegrass, and Kentucky bluegrass, respectively (Fig. 7d-f).

Fig. 7
figure 7

Effects of elevated CO2 concentrations on the relationships between Vcmax and leaf N (a-c) or Jmax and leaf N (d-f) for the three grass species. Values given are mean ± standard deviation for n = 4 pots

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Discussion

Different optimal CO2 fertilization concentrations for the growth of perennial grasses

Most plants generally benefit from elevated atmospheric CO2 concentration through the “CO2 fertilization effect”, which boosts growth and yield [19, 23, 46, 52]. However, this positive CO2 fertilization effect strongly depends on the plant functional groups and species [7, 22, 53,54,55,56]. Even within the same species of winter wheat, the results from previous studies are inconsistent [22, 50, 57,58,59,60]. These contradictory results suggest that different plants and/or species may have different optimal CO2 concentrations for their growth. Our results showed that the optimal CO2 concentrations occurred at 945, 915, and 1151 ppm for the aboveground biomass and at 915, 1178, and 1386 ppm for the total biomass of tall fescue, perennial ryegrass, and Kentucky bluegrass (Fig. 1), suggesting that a strong CO2 fertilization effect occurred at different optimal CO2 concentrations for these three perennial grasses. This result also indicated that Kentucky bluegrass has the highest optimal CO2 concentration among the three grasses, and thus may suffer less from future climate change than the other two grasses. In addition, by enhancing the atmospheric CO2 concentration from 400 ppm to the optimum for each grass species, the maximum CO2 fertilization effect substantially increased the total biomass of the by 60%, 15%, and 30% for tall fescue, perennial ryegrass, and Kentucky bluegrass respectively. Interestingly, biomass enhancements of 15% and 30% for perennial ryegrass and Kentucky bluegrass are very similar with the average of approximately 20% for C3 plants as estimated in meta-analysis of Free-Air CO2 Enrichment (FACE) studies [61, 62], and 32% of Open Top Chamber (OTC) and greenhouse experiments [63]. However, the increased rate of tall fescue (60%) is much higher than those of the other two species, indicating this specie will benefit the most from the positive fertilization effect among these three perennial grasses under future high CO2 environmental conditions. It is noted that we found no obviously optimal CO2 for the belowground biomass of two species (Kentucky bluegrass and perennial ryegrass), as evidenced by the upward quadratic relationships between belowground biomass and CO2 concentrations. These results suggest that the carbon allocation between aboveground and belowground of the three grasses characterize different strategies, and tall fescue might select a more effective strategy to balance the carbon investment between aboveground and belowground than the other two species under high CO2 concentrations.

The positive CO2 fertilization effect on the growth of perennial grasses

Previous studies have well demonstrated that plant growth is highly correlated with biochemical and photochemical processes [64, 65] such as photosynthesis and respiration, through which the CO2 fertilization effect is developed and regulated [22]. In the current study, the photosynthesis-CO2 relationship followed a similar bell-shaped curve like the biomass-CO2 relationship (Figs. 1 and 2), suggesting that the positive CO2 fertilization effect might be attributed to the up-regulation of An, as evidenced by the increased leaf net photosynthetic rates (An), with the maximum CO2 fertilization effect occurring at 959 ppm for Kentucky bluegrass, and 1200 ppm for both tall fescue and perennial ryegrass (Fig. 2a). Further analysis showed that leaf biochemical and photochemical processes played a key role in determining the positive CO2 fertilization effect through directly increasing both carboxylation rates and electron transport rates of perennial grasses. Our results showed that both the maximum carboxylation rate of Rubisco (Vcmax) and the maximum capacity of electron transport RuBP regeneration (Jmax) of the three grasses were dramatically stimulated by elevated CO2 concentrations before reaching their optimums (Fig. 3), suggesting that the initial increase in CO2 concentration may favor both the light and dark reactions of photosynthesis through boosting the Rubisco carboxylation and the RuBP regeneration processes. Also, a recent study has reported that the Vcmax of winter wheat was dramatically increased by elevating ambient CO2 concentrations from 400 ppm to about 800 ppm [23].

In addition to leaf photosynthesis, the positive CO2 fertilization effect on the growth of perennial grasses may also closely associate with the changes in leaf respiration and total non-structural carbohydrates (TNC) under high CO2 concentrations. Our results showed that the leaf dark respiratory rates (Rd) and leaf TNC of the three grasses consistently decreased with elevated CO2 concentrations. Meanwhile, we found a linear relationship between leaf Rd and TNC, suggesting that Rd reduction may partially attribute to decrease in leaf TNC, which is the most important substrate for leaf respiration [14, 66]. Overall, the up-regulation of An and the decline of Rd may both play pivotal roles in explaining the positive CO2 fertilization effects on the growth of perennial grasses in the current study.

The diminishing returns of CO2 fertilization effect on perennial grasses

Previous studies have found that beyond certain thresholds, high CO2 concentration cause diminishing returns of CO2 fertilization effect on plants [13, 22, 23]. Several studies found that the stimulation of An induced by elevated CO2 decreased or even diminished if exposed for a longer time period, because plants acclimate to elevated CO2 concentrations through a process known as down-regulation [19, 32]. We also found bell-shaped curves for biomass-CO2 relationships for the three grasses similar to the An-CO2, indicating a reduction in biomass due to a decline in the photosynthetic rate at high CO2 concentrations. It is well demonstrated that the down-regulation of An is possibly attributed to the changes in carbohydrates [31], under high CO2 environments. In the current study, elevated CO2 concentrations beyond the optima of the three grasses consistently reduced leaf TNC, suggesting that the imbalance of carbohydrate concentration in the source and sink was not a limiting factor for the down-regulation of An. In addition, it is important to noted that hexokinase is a key functional enzyme for mediating sugar sensing [67] and may also decrease Rubisco content through inhibiting the expression of photosynthetic genes [68]. Previous studies have well demonstrated that the Rubisco content and activity of higher plants were dramatically decreased under high CO2 concentrations [68, 69], because leaf N was prior to enzymes relating to the metabolic processes of starch and sucrose than invested in Rubisco when plants was subjected to high CO2 concentrations [70]. Consequently, the changes in hexokinase with CO2 concentrations may contribute to the bell-shaped relationship between An and CO2 concentration, especially for the down-regulation of An under high CO2 concentrations.

It is well documented that stomatal conductance (gs) declines when exposed to elevated atmospheric CO2 concentration, and a doubling of CO2 from the present ambient concentration generally results in a reduction in gs of 10–70% depending on species or functional groups [58]. In the current study, we also found that the gs of all three grasses were dramatically decreased with elevated CO2 concentrations, which may be partly due to the down-regulation of An caused by CO2. Moreover, the reduced gs under high CO2 concentrations might result in a decline in leaf transpiration and thus reduced nutrient availability, as observed in many previous studies [22]. Previous studies have claimed that elevated CO2 concentration increased plant C/N ratios mainly due to a decrease in N content [12, 26]. Similarly, we also found that the nitrogen contents of both tall fescue and Kentucky bluegrass were markedly decreased with increasing CO2 concentrations, which may also be caused by the CO2 effects on An, since nitrogen content is associated with photosynthetic enzymes such as Rubisco [35,36,37]. In addition, the linearly positive relationships between leaf N and Vcmax for the three grasses (Fig. 7) were directly supporting the above conclusion that the down-regulation of An was partly attributed to the decline of leaf N under high CO2 concentrations.

It should be noted that the CO2 fertilization effect on plant growth may be confounded by future climate change such as global warming, nitrogen deposition, and drought, which may reduce or cancel out the CO2 fertilization effect [39]. For example, the global surface temperature may continue to increase and cause global precipitation to become unevenly distributed both temporally and spatially [2]. As a result, drought stress caused by the increased global surface temperature and the declined precipitation may also be a critical factor affecting leaf photosynthesis and respiration [17] and thus plant growth and biomass accumulation [49], and in turn the structure and function of ecosystems such as grasslands and pastures [37, 40]. Therefore, the fates of the three grasses cannot only be determined by elevated CO2 concentrations because warming and drought may have interactive effects with CO2 enhancement on the growth, physiological, and biological processes of the three grasses under future climate change [20]. Therefore, more controlled experiments with multiple factors such as temperature, drought, nutrition availability and CO2 concentration are needed for predicting the fates of grass species and thus the community dynamics of grasslands under future global climate change [31]. However, it is important to note that this study was carried out under controlled conditions with sufficient nutrients and water for plants during the experiment, which is obviously different from actual field conditions. Therefore, many similar experiments should be carried out in natural conditions without fertilization and watering for predicting the fates of the three cool-season C3 grasses in future climate change scenarios.

Conclusions

We found that the optimal CO2 concentrations occurred at 945, 915, and 1151 ppm for the aboveground biomass of tall fescue, perennial ryegrass, and Kentucky bluegrass, respectively. Higher CO2 concentrations had diminishing returns of CO2 fertilization effect on plant growth, causing limiting effects on stomatal conductance, nitrogen availability and changes in the biochemical and photochemical processes of photosynthesis. Our results suggest that the continuously increasing atmospheric CO2 concentration in the future may dramatically lower the CO2 fertilization effect, and thus many current climate change models based on earlier results of “doubling–CO2” experiments may overestimate the CO2 fertilization effect on grasslands beyond the optimum CO2 concentration. According to recent IPCC reports, if global CO2 emissions are not effectively mitigated, the atmospheric CO2 concentration might be over 900 ppm in the second half of this Century. Nevertheless, the optimal CO2 concentrations found in this study can be used as an indicator in predicting the fates of the cool-season C3 grasses under future rising atmospheric CO2 concentration and climate change, because grasses with high optimal CO2 concentrations may take full advantage of the CO2 fertilization effect.