Effects of Elevated Carbon Dioxide and Chronic Warming on Nitrogen (N)-Uptake Rate, -Assimilation, and -Concentration of Wheat

The concentration of nitrogen (N) in vegetative tissues is largely dependent on the balance among growth, root N uptake, and N assimilation. Elevated CO2 (eCO2) plus warming is likely to affect the vegetative-tissue N and protein concentration of wheat by altering N metabolism, but this is poorly understood. To investigate this, spring wheat (Triticum aestivum) was grown for three weeks at two levels of CO2 (400 or 700 ppm) and two temperature regimes (26/21 or 31/26 °C, day/night). Plant dry mass, plant %N, protein concentrations, NO3− and NH4+ root uptake rates (using 15NO3 or 15NH4), and whole-plant N- and NO3--assimilation were measured. Plant growth, %N, protein concentration, and root N-uptake rate were each significantly affected only by CO2, while N- and NO3−-assimilation were significantly affected only by temperature. However, plants grown at eCO2 plus warming had the lowest concentrations of N and protein. These results suggest that one strategy breeding programs can implement to minimize the negative effects of eCO2 and warming on wheat tissue N would be to target the maintenance of root N uptake rate at eCO2 and N assimilation at higher growth temperatures.


Introduction
Wheat (Triticum aestivum) ranks third among field crops globally and in the United States (U.S.) in terms of production, behind corn (Zea mays) and rice (Oryza sativa) globally or corn and soybean (Glycine max) nationally [1,2]. Wheat grain protein content is a major determinant of baking quality and it largely depends on the nitrogen (N) concentration during the vegetative stage of growth because the grains receive most of their N from vegetative tissues via remobilization [3,4]. Though tissue N can be enhanced by improved N fertilization, the drawbacks of using more N fertilizer to boost N content include higher costs of production and environmental problems caused by excess application of N fertilizers. The concentration of N in vegetative tissues is largely dependent on the balance among growth, root N uptake, and N assimilation [5,6]. Climate change will impact plant growth and N metabolism, but these impacts have mostly been studied to date by examining the effects of individual climate-change factors (especially eCO 2 , higher temperatures, and drought). However, since these main climate-change factors will change concomitantly, discerning the interactive effects of these factors will be necessary to understand how climate change will impact plant N metabolism.
Many studies have investigated the individual effects of eCO 2 or warming on plant N relations, but data pertaining to the interactive effects of these two variables on N relations are scarce. To illustrate, Plants 2020, 9,1689 2 of 10 the effects of eCO 2 plus warming on tissue N and protein concentrations of wheat and other species are very limited. Studies have shown that eCO 2 plus warming can reduce leaf N concentration of wheat [7,8]. Most past studies that have investigated the interactive effects of eCO 2 and warming on tissue N concentration of species other than wheat found a non-significant effect on root N concentration [9][10][11][12][13][14], but a trend towards decreasing shoot or leaf N concentration [9,[15][16][17]. However, there are reports that show that eCO 2 plus warming can either increase or decrease root N concentration [11,13,17], while effects on the shoot or leaf N concentration can be positive [8,10] or neutral [10,12,18,19]. Collectively, these studies indicate that the response of plant N concentration to eCO 2 plus warming can be variable, likely due in part to differences in experimental protocols and plant species.
Effects of eCO 2 plus warming on the vegetative tissue protein concentration of wheat has scarcely been studied. A study conducted by Jauregui et al. (2015) [7] found a negative effect of eCO 2 plus warming on flag-leaf total-soluble protein concentrations of wheat. None of the previous studies investigated eCO 2 plus warming effects on whole-plant protein concentration of wheat. Previously, Jayawardena et al. (2017) [10] examined the effects of eCO 2 plus warming on the total root protein concentration of tomato (Solanum lycopersicum) provided either NO 3 − or NH 4 + as the sole N source, and they noted significant decreases of root protein concentration in both sets of plants in response to eCO 2 plus warming. In addition, the effects of eCO 2 plus warming on plant N uptake have rarely been studied. The two studies that examined this in wheat measured N uptake as the amount of N in above-ground organs (g m −2 ), without taking root N into account, and both studies found no interactive effect of eCO 2 and temperature on wheat N uptake [8,20]. In contrast, research from other species suggests an inconsistent effect of N-uptake rate in response to eCO 2 plus warming. For example, a growth chamber study showed that eCO 2 (700 vs. 400 ppm) plus warming (38 vs. 28 • C) reduced N-uptake rate of the C 3 species Abutilon theophrasti, but they saw varying N-uptake rates with the C 4 species Amaranthus retroflexus at different plant growth stages [21]. Meanwhile, no significant effect of eCO 2 (510 ppm vs. ambient) plus warming on both NO 3 − and NH 4 + -uptake rates of Calluna vulgaris and Deschampsia flexuosa was observed [22]. Using sequential harvesting, we previously examined the N-uptake rate of S. lycopersicum in response to CO 2 (700 vs. 400 ppm) plus warming (37 vs. 30 • C or 38 vs. 33 • C) and discovered that eCO 2 plus warming inhibited N-uptake rate [10]. As with N-uptake rate, N assimilation in response to eCO 2 plus warming has rarely been studied, and collective results from the few studies suggest a tendency for N assimilation to decrease in response to eCO 2 plus warming. Based on a gene-expression analysis in Triticum durum, eCO 2 (700 vs. 370 ppm) plus warming (ambient + 4 • C) may inhibit N assimilation [23]. Similarly, a greenhouse experiment also reported that eCO 2 (700 vs. 400 ppm) plus warming (ambient + 4 • C) inhibited N assimilation in flag leaves of T. durum [7]. In a previous study, based on root %N and protein data of S. lycopersicum, we suggested that eCO 2 (700 vs. 400 ppm) plus warming (37 vs. 30 • C) inhibited root N assimilation [10].
The above-mentioned studies demonstrate that the effects of eCO 2 plus warming on plant N metabolism are poorly understood, especially in wheat. A better understanding of wheat N metabolism in response to predicted future climate conditions is essential to improve the N-use efficiency of wheat. Therefore, the objective of this study was to determine the individual and interactive effects of eCO 2 and chronic warming on plant growth, root NO 3 − and NH 4 + -uptake rates, whole-plant N-and NO 3 − -assimilation, and whole-plant protein concentration of wheat. Results of this study will help to plant breeders to develop new wheat cultivars better adapted to future climate conditions.
Plants 2020, 11, x FOR PEER REVIEW 3 of 10 NH4 15 NO3 and 15 NH4NO3-supplied plants (i.e., in two independent sets of plants harvested two days apart) (Table S1, Figure 1). Across both temperatures, eCO2 significantly decreased plant %N (except 15 NO3 − supplied plants at 26 °C), while chronic warming did not influence %N (Figure 2A,B). Notably, %N was lowest in plants grown at eCO2 plus warming. Across both temperatures, eCO2 decreased NO3 − -uptake rate (more so at 26 °C) and NH4 + -uptake rate ( Figure 2C,D). In contrast, chronic warming did not affect NO3 − -uptake rate but did marginally increase NH4 + -uptake rates ( Figure 2C,D). Among all treatment combinations, NH4 + -uptake rates were consistently greater than NO3 − -uptake rates.  The ratios of total-plant inorganic N:total N and total-plant NO 3 − :total N were significantly affected only by temperature (Table S1). Elevated CO 2 non-significantly increased the inorganic N:total Plants 2020, 9, 1689 4 of 10 N and NO 3 − :total N ratios at 26 • C, while decreasing the ratios at 31 • C ( Figure 3). In contrast, chronic warming significantly or non-significantly increased the inorganic N:total N and NO 3 − :total N ratios across CO 2 levels.
Plants 2020, 11, x FOR PEER REVIEW 4 of 10 The ratios of total-plant inorganic N:total N and total-plant NO3 − :total N were significantly affected only by temperature (Table S1). Elevated CO2 non-significantly increased the inorganic N:total N and NO3 − :total N ratios at 26 °C, while decreasing the ratios at 31 °C ( Figure 3). In contrast, chronic warming significantly or non-significantly increased the inorganic N:total N and NO3 − :total N ratios across CO2 levels. The whole-plant total-protein concentration was significantly reduced only by CO2 (Table S1, Figure 4).  The whole-plant total-protein concentration was significantly reduced only by CO 2 (Table S1, Figure 4). The ratios of total-plant inorganic N:total N and total-plant NO3 − :total N were significantly affected only by temperature (Table S1). Elevated CO2 non-significantly increased the inorganic N:total N and NO3 − :total N ratios at 26 °C, while decreasing the ratios at 31 °C (Figure 3). In contrast, chronic warming significantly or non-significantly increased the inorganic N:total N and NO3 − :total N ratios across CO2 levels. The whole-plant total-protein concentration was significantly reduced only by CO2 (Table S1, Figure 4).

Discussion
In the present study, eCO 2 enhanced the growth of wheat irrespective of the temperature. In contrast, though statistically not significant, warming caused a slight decrease in plant growth at each CO 2 level. Previously, we noticed severe inhibition of tomato growth caused by the combination of eCO 2 and warming [10,24], which was partly due to a dramatic increase in leaf angle, and thus decrease in photosynthesis, compared to eCO 2 or warming alone [24]. This growth response was not observed in wheat. These results indicate that interactive effects of CO 2 enrichment and warming will be species-specific, and not necessarily additive as observed in tomato.
Typically, plants grown under eCO 2 have lower tissue N concentrations due to increased photosynthetic assimilation of C and reduced N uptake caused by various reasons, such as the closure of stomates, down-regulation of Ribulose-1,5-bisphosphate carboxylase/oxygenase (RUBISCO), and increased lateral roots-to-primary roots ratio [6]. Similarly, in this study, wheat plants grown under eCO 2 had lower plant %N regardless of the temperature treatment; moreover, plants grown at eCO 2 plus warming had the lowest plant %N. Elevated CO 2 -driven growth stimulation and decreased %N indicate that plants cannot increase their N-uptake rate to keep pace with enhanced plant growth under eCO 2 . In fact, both NO 3 − and NH 4 + -uptake rates decreased slightly with eCO 2 at 26 • C; however, both NO 3 − and NH 4 + -uptake rates were unaffected by eCO 2 at 31 • C. These results indicate that the plants grown at eCO 2 plus warming were able to maintain N-uptake rate per unit root, yet tissue N concentration in these plants still decreased, which could be due to growth dilution. Meanwhile, chronic warming at ambient CO 2 (aCO 2 ) caused a slight increase in plant %N, and it was correlated with a marginal increase in NH 4 + -uptake rate. Although the NO 3 − -uptake rate is favored over NH 4 + -uptake rate by rising soil or root temperatures in some species [25], in the present study, wheat NO 3 − -uptake rate was unaffected by warming treatment.
In this study, the ratios of inorganic N:total N and NO 3 -:total N were used as indices for whole-plant N-and NO 3 − -assimilation, respectively. Chronic warming increased both inorganic N:total N and NO 3 − :total N ratios, irrespective of the CO 2 treatment, suggesting inhibition of both N and NO 3 − assimilation by warming. In addition, the magnitude of warming-driven inhibition was partially offset by eCO 2 . Based on these results, we expected plants grown at eCO 2 plus 31 • C to have low protein levels, and plants grown at aCO 2 plus 31 • C to have the lowest protein levels. However, the protein data were in partial disagreement with these predictions. As expected from inorganic N:total N and NO 3 − :total N ratios, plants grown at eCO 2 plus warming had the lowest protein concentration, which suggests that the low protein levels were due to the inhibition of N and NO 3 − assimilation by eCO 2 plus warming. Previously, Jauregui et al. (2015) [7] reported inhibition of leaf N assimilation in T. durum grown at eCO 2 plus warming, and they proposed that the inhibition could be due to decreased leaf photorespiration and dark respiration. However, in this study, plants grown at aCO 2 plus warming had the highest protein concentration, as well as the highest ratios of inorganic N:total N and NO 3 − :total N among all treatment combinations, indicating an accumulation of inorganic N forms.
Though statistically not significant, eCO 2 tended to decrease N-and NO 3 − -assimilation at 26 • C, and, as a result, plants grown at eCO 2 plus 26 • C tended to have low protein concentrations. In summary, eCO 2 reduced wheat tissue N and protein concentrations regardless of the temperature treatment, but the magnitude of decrease tended to be greater when combined with warming. This decrease was not due to the decreased N-uptake rate, since both NO 3 − and NH 4 + -uptake rates were not lowest in the eCO 2 plus warming treatment. As ratios of inorganic N:total N and NO 3 − :total N suggest, low protein levels at eCO 2 plus warming were likely due to the inhibition of N assimilation. To date, eCO 2 plus warming effects on yield components of wheat have been studied in some detail [8,20,26,27]. In the present study, we investigated the N relations of wheat when plants were in the vegetative stage. Wheat plants reach their maximum N content when they are nearing anthesis [28], and about 50-90% of the final grain N is accumulated before anthesis and later remobilized from vegetative tissues to grains [3,4,29]. Therefore, the changes in N relations at the vegetative stage that we observed are likely to have a direct impact on determining final wheat grain quality. Together, these results indicate that crop improvement strategies, such as transgenic approaches, genetic engineering, and traditional plant breeding, might focus on developing wheat cultivars which can maintain root N uptake rate at eCO 2 and N assimilation at higher growth temperatures in order to enhance the nutritional quality of wheat in a warming world enriched with CO 2 .

Plant Material, Growth Conditions, and Treatments
Wheat (T. aestivum L. cv. Glenn), which is a cool-season C 3 grass, was used as the model species. It is a hard-red spring wheat cultivar developed by North Dakota State University (NDSU, Fargo, ND, USA) for high yields, high protein, and scab resistance, and it was released by the North Dakota Agricultural Experiment Station in 2005 [30].
Seeds were sown in trays filled with calcined clay in a greenhouse and watered daily. Air temperature in the greenhouse fluctuated between 25-30 • C. When photosynthetically active radiation (PAR) dropped below 300 µmol m −2 s −1 , 250-W high-pressure sodium (GE Lighting Inc., Cleveland, OH, USA) and 400-W metal-halide (Osram Sylvania Products Inc., Manchester, NH, USA) lamps provided supplementary lighting to maintain a 15-h photoperiod. When seedlings were 12-cm tall, 44 seedlings having two leaves were transplanted into 3.1 L cylindrical pots (10-cm diameter × 40-cm length PVC pipes; one plant per pot) containing a mixture of calcined clay and perlite in a 3:1 (v:v) ratio and supported by mesh at the bottom of the pot.
Pots were transferred to four growth chambers (model E36HO, Percival Scientific Inc., Perry, IA, USA), with 11 pots per chamber. A 2 × 2 factorial experimental design was used, with CO 2 levels of ambient (400 ppm) vs. elevated (700 ppm) and temperatures of near-optimal (26/21 • C; day/night) vs. chronic warming (31/26 • C; day/night). According to the low and intermediate CO 2 emission scenarios, atmospheric CO 2 is likely to be between 450-1000 ppm by 2100 [31]. Therefore, we chose 700 ppm as our eCO 2 treatment, which is an intermediate value between today's CO 2 level and the upper limit of the intermediate CO 2 emission scenario. Near-optimal and supra-optimal temperatures were chosen based on preliminary experiments. Plants were acclimated to the new chamber environment for four days at 600 µmol m −2 s −1 PAR (supplied by 55-W Osram Dulux luminous lamps; Osram GmbH, Augsburg, Germany) with a 14-h (0600-2000 HR) photoperiod, 26/21 • C (day/night near-optimum growth temperatures), ambient CO 2 (400 ppm), and ambient humidity. During this period, 500 mL of quarter-strength nutrient solution was added to each pot twice (nutrient concentrations of the full-strength solution: 2 mM MgSO 4 , 1 mM KH 2 PO 4 , 1 mM K 2 HPO 4 , 2 mM CaCl 2 , 71 µM Fe-DTPA, 10 µM MnCl 2 , 50 µM H 3 BO 3 , 6 µM CuSO 4 , 6 µM ZnSO 4 , 1 µM Na 2 MoO 4 , 1 mM NH 4 NO 3 ; pH = 6.0). When plants were free from transplant-stress, the temperature of the high-temperature-treatment chambers was gradually increased from 26 • C to 31 • C over three days to avoid potential heat-shock. Once high-temperature-treatment chambers reached 31 • C, CO 2 treatments were started (day 0). Plants were fertilized with 500 mL of full-strength complete nutrient solution every other day. They were rotated within chambers every 4-5 days to avoid potential position effects and switched between chambers every 7-8 days to avoid potential chamber effects.
The nutrient solution with stable isotopes was made by adding either NH 4 15 NO 3 or 15 NH 4 NO 3 with an isotopic purity of 98 atom% 15 Figure S1). When harvesting, plants were at their stem elongation stage before booting.

Plant N and Protein Analysis
Harvested plants were split into shoots and roots. Roots were cleaned with tap water. Each root and shoot system was longitudinally divided into two halves using a scissor and weighed separately to the nearest 0.001 gram. One half from each was flash-frozen in liquid N 2 and stored at −80 • C to be used for protein and NH 4 + quantification (fresh tissue Briefly, 50 mg of ground dried tissue was suspended in 5 mL of de-ionized water. After incubation at 45 • C for 1 h and centrifugation (model 5810R, Eppendorf, Framingham, MA, USA) at 10,000 rpm for 5 min at room temperature, the supernatant was recovered. Then, 0.2 mL of recovered supernatant was reacted with 0.8 mL of 5% (w/v) salicylic acid-sulfuric solution and 19 mL of 2N NaOH. Nitrate was quantified by measuring absorbance (model UV-1650-PC, Shimadzu, Columbia, MD, USA) at 410 nm using KNO 3 standards with concentration ranging from 10-60 µg mL −1 . When quantifying NH 4 + , 500 mg of fresh tissue was ground into a fine powder using liquid N 2 . Then, NH 4 + was extracted into 30 mL of 0.001M acidic CaSO 4 solution (pH = 3) and quantified using an NH 4 + ion-selective electrode (Hach company, Loveland, CO, USA) using two technical replicates for each biological replicate. The total plant inorganic N content was estimated as the sum of total-NO 3 − and -NH 4 + contents with which the ratios of total inorganic N:total N and total NO 3 − :total N were calculated.
Shoot and root proteins were extracted as described in Jayawardena et al. (2017) [10]. Briefly, 1 g of fresh tissue was ground to a fine powder with liquid N 2 and then with 4 mL of an extraction buffer [0.2 M Tris-HCl, pH = 8.0; 5 mM ethylenediaminetetraacetic acid (EDTA), pH = 7.5-8.0; 0.7 M sucrose; 1% sodium dodecyl sulfate (SDS); 1 mM phenylmethylsulfonyl fluoride; 1 mM leupeptin; and 2% β-mercaptoethanol]. Protein was extracted into a phenol phase by adding an equal volume of buffer-saturated phenol (pH = 6.6-8.0) following centrifugation (10,000 rpm for 10 min at 4 • C). Extracted protein was pelleted down by centrifugation (10,000 rpm for 10 min at 4 • C) after adding five volumes of 0.1 M ammonium acetate in methanol. The protein pellet was washed several times with ammonium acetate and 100% acetone. Then, the protein pellet was air-dried within less than a min under room temperature. Each root and shoot protein pellet was added to 1.6 mL and 3 mL of a re-solubilizing buffer (62.5 mM Tris-HCl, pH = 6.8; 0.5 SDS; 20% glycerol), respectively, to dissolve them. Protein concentration was determined by measuring absorbance (model UV-1650-PC, Shimadzu) at 750 nm using a colorimetric assay (DC protein assay; Bio-Rad Laboratories Inc., Hercules, CA, USA) and bovine serum albumin (BSA) as the protein standard. Two and three technical replicates were used for each unknown and standard sample, respectively.

Statistical analysis
Data were analyzed using RStudio version 3.6.1 (RCore Team, Vienna, Austria). Statistical assumptions of independence, normality and equal variance were checked with residual