1. Introduction
Anthropogenic CO
2 and other greenhouse gas emissions have increased significantly since industrialization, warming the planet. Global climate models predict atmospheric CO
2 concentration will be about 420 to 935 ppm, and global mean surface temperature will increase by about 1.4 to 5.8 °C, by the end of this century [
1,
2]. This rise in temperature may cause both acute and chronic heat stress in plants, affecting both root and shoot functions [
3]. Although CO
2 enrichment alone benefits plants (e.g., increased photosynthesis and water-use efficiency), these beneficial effects may disappear when eCO
2 is compounded with other climate-change variables, such as supra-optimal temperatures [
4,
5,
6,
7]. For example, the combination of eCO
2 and warming decreased growth and root N-uptake rate in tomato, relative to either factor alone [
5]. In addition, eCO
2 alone is not always beneficial to plants, as it can result in a dilution of tissue N concentration (%N) due to increased photosynthetic assimilation of C, resulting in plant tissue of lower nutritional quality for food [
8,
9].
Since plants procure and assimilate nutrients through nutrient-specific-uptake and -assimilatory proteins, crop improvement under future climate conditions could be achieved by modification of these proteins using transgenic or genetic engineering, or traditional plant breeding approaches, the latter by identifying species or genotypes with better-adapted nutrient-uptake and -assimilation mechanisms [
8]. However, in order to implement such efforts, we should identify which biochemical pathway (e.g., uptake vs. translocation vs. assimilation) and which biochemical component (e.g., uptake proteins vs. assimilatory proteins) to target. Nitrogen is often the most limiting nutrient for plants [
10]. Plants can procure N either in inorganic or organic forms, but most plants procure the majority of their N primarily as inorganic N (NO
3− and NH
4+) [
11]. A number of studies have investigated the effects of eCO
2, or warming alone, on plant N uptake, N translocation, and N assimilation, but studies examining the combined effects of eCO
2 and warming on these responses are scarce.
Though eCO
2 tends to decrease root N-uptake rate (N uptake per unit mass or length), past studies show that root N-uptake rate in response to eCO
2 can be highly variable and can depend on the form of N supplied [
9,
12,
13]. Optimum temperature for plant growth and function is species specific, and the effect of warming on root N uptake depends on whether the temperature increase is from suboptimal to optimal or from optimal to supra-optimal. Warming from suboptimal to optimal often increases root N-uptake rate [
14,
15,
16,
17], while warming from optimal to supra-optimal (acute or chronic) often decreases root N uptake [
17,
18,
19,
20,
21]. The limited evidence available suggests that the interactive effects of eCO
2 and warming on N uptake can be equivocal. Coleman and Bazzaz [
22], using
Abutilon theophrasti, and Jayawardena et al. [
5], using
Solanum lycopersicum, showed that the interactive effect of eCO
2 plus warming can inhibit root N-uptake rate in C
3 plants when compared with other treatments (i.e., eCO
2 or warming alone). In addition, in the C
4 species,
Amaranthus retroflexus, N-uptake rates varied with plant ages in response to eCO
2 plus warming [
22]. Using
15N labeling, Arndal et al. [
23] found no effect of eCO
2 plus warming on NO
3− nor NH
4+-uptake rates of
Calluna vulgaris (an evergreen dwarf shrub) and
Deschampsia flexuosa (a perennial grass). Dijkstra et al. [
24] also found no interactive effect of eCO
2 and warming on NO
3− uptake of grasses in a semiarid grassland.
Studies that investigated root-to-shoot N translocation in response to eCO
2 showed or suggested a consistent decreasing trend with eCO
2 [
25,
26,
27,
28,
29]. As Cohen et al. [
26] explained, one potential reason for decreased N translocation in response to eCO
2 could be the reduced size of xylem volume when plants are grown at eCO
2. Nitrogen translocation from roots to shoots in response to temperature has been studied in some detail, but results were highly variable. In most studies, the highest temperature examined was 30 °C or less, and the temperature was altered only in the root-zone while maintaining the shoots at a control temperature [
15,
30,
31,
32,
33,
34]. These studies showed that increased root-zone temperature can increase [
15,
31,
32], decrease [
32,
34], or have no effect [
33] on, root-to-shoot N translocation. Moreover, a study conducted by Hungria and Kaschuk [
35] showed that whole-plant heat stress (39 vs. 28 or 34 °C) can reduce xylem organic-N translocation in
Phaseolus vulgaris, while Mainali et al. [
21] suggested whole-plant acute heat stress (40 vs. 30 °C) did not affect N translocation in
Andropogon geradii. As with N-uptake rate, data on root-to-shoot N translocation in response to eCO
2 plus warming are scarce. Rufty et al. [
31] studied the interactive effect of root-zone temperature (18, 24, and 30 °C) and eCO
2 (1000 vs. 400 ppm) on N translocation of
Glycine max supplied NO
3− as the sole N source, and they noted an increase with eCO
2 plus root warming. In contrast, based on lower transpiration and leaf
15N isotopic composition observed in
Triticum durum at eCO
2 (700 vs. 400 ppm) plus warming (ambient vs. ambient + 4 °C), Jauregui et al. [
4] concluded that eCO
2 plus warming can reduce root-to-shoot N translocation.
Plant N assimilation in response to eCO
2 has been extensively studied. A number of studies have shown that eCO
2 can inhibit shoot NO
3−, but not NH
4+, assimilation in C
3 plants [
36,
37,
38,
39]. However, challenging this view, Andrews et al. [
40] showed that eCO
2 does not inhibit NO
3− assimilation in C
3 plants, and the assimilation of both forms of N take place in a similar way in response to eCO
2. In response, Bloom et al. [
41] stated that eCO
2 inhibits shoot NO
3− assimilation, but enhances NO
3− assimilation in roots of C
3 plants. This is consistent with results of Jauregui et al. [
27], who suggested that eCO
2 favored N assimilation in roots over shoots in
T. durum, based on the low shoot-to-root NR activity ratio observed at eCO
2. As with N uptake, one could expect N assimilation to increase as temperature rises from suboptimal-to-optimal, and decrease with optimal-to-supra-optimal temperatures, because N assimilation is carried out by enzymes which have temperature optima. The majority of studies that have investigated temperature effects on shoot N assimilation have looked at the effect of heat stress on NR activity only, and they reveal that NR activity diminished with heat stress [
35,
42,
43,
44]. Using two
Agrostis species, Rachmilevitch et al. [
45] investigated the effects of root-zone temperature (37 vs. 20 °C) on the rate of plant NO
3− assimilation, and noted it decreased with increased temperature. The limited evidence from these studies suggests that optimal to supra-optimal temperature increases are most likely to reduce plant N or NO
3− assimilation. Nitrogen assimilation is a key process that influences the nutritional quality of food and, recently, researchers have started investigating how it responds to eCO
2 plus warming. To date, three reports showed a decreasing trend for N assimilation in response to eCO
2 plus warming. Vicente et al. [
46] investigated the effects of eCO
2 (700 vs. 370 ppm) and warming (ambient vs. ambient + 4 °C) at two levels of N supply on C and N metabolism of
T. durum, using gene expression analysis. Based on decreased soluble protein, amino acids, and NR activity in flag leaves, they showed that N assimilation can be inhibited by eCO
2 and warming. Since most of the genes involved in N metabolism are post-transcriptionally or post-translationally regulated [
10,
47], gene expression measurements alone do not necessarily reflect phenotypic effects on N metabolism. A study conducted by Jauregui et al. [
4] also reported that eCO
2 (700 vs. 400 ppm) plus warming (ambient + 4 °C) inhibited N assimilation in flag leaves of
T. durum, based on decreased levels of amino acids, total soluble protein, and NR activity. Root N assimilation in response to eCO
2 (700 vs. 400 ppm) plus chronic warming (37 vs. 30 °C) was indirectly investigated by Jayawardena et al. [
5] in
S. lycopersicum, and they suggested eCO
2 plus warming can inhibit root N assimilation, which could have resulted from the observed decreases in levels of N-assimilatory proteins that were measured in roots. Notably, none of the previous studies investigated whole-plant N assimilation in response to eCO
2 plus warming.
The aforementioned review reveals a lack of studies have investigated the combined influence of eCO2 and chronic warming on plant N metabolism. Therefore, the objective of this study was to determine the individual and interactive effects of eCO2 and chronic warming on NO3− and NH4+ uptake rates, net N translocation, and whole-plant N and NO3− assimilation, using tomato (S. lycopersicum) as a model. The information resulting from this study will be helpful for crop scientists, plant breeders, and molecular biologists to understand how N metabolism of tomato and other plants will respond to future climates, and how to develop new tomato genotypes with improved N use under future climate conditions.
2. Results
Total plant dry mass was significantly decreased with chronic warming (
Table S1,
Figure 1). In contrast, the effect of eCO
2 on plant dry mass was dependent on the treatment temperature. Elevated CO
2 significantly and non-significantly increased the plant dry mass of
15NH
4+-supplied and
15NO
3−-supplied plants at 33 °C, respectively, while it significantly decreased the plant dry mass of both sets of plants at 38 °C. Plants grown at eCO
2 plus chronic warming had the lowest dry mass.
Both NO
3− and NH
4+-uptake rates were significantly affected by the interaction of CO
2 × temperature (
Table S1). Though chronic warming significantly increased NO
3−-uptake rate at ambient CO
2 (aCO
2), it did not influence NH
4+-uptake rate at aCO
2. Elevated CO
2 did not influence either NO
3− or NH
4+-uptake rates at 33 °C, but it tended to decrease NO
3−-uptake rate and significantly decreased NH
4+-uptake rate at 38 °C (relative to 33 °C and aCO
2). Hence, as with total plant dry mass, plants grown at eCO
2 plus chronic warming had the lowest NO
3− and NH
4+-uptake rates (
Figure 2). Notably, the NH
4+-uptake rate was greater than the NO
3−-uptake rate at 33 °C, regardless of the CO
2 treatment (approximately ×1.4). However, when the temperature increased from 33 °C to 38 °C, NO
3−-uptake rate surpassed the NH
4+-uptake rate, regardless of the CO
2 treatment (approximately ×1.1–1.2).
The ratio of total
15N content in the shoots vs. roots of
15NO
3−-supplied plants was significantly affected only by the temperature, while that of
15NH
4+-supplied plants was significantly affected by both temperature and the interaction of CO
2 × temperature (
Table S1). Chronic warming tended to decrease the ratio of total
15N content in the shoots vs. roots of
15NO
3−-supplied plants at aCO
2. However, it did not influence the ratio of
15NH
4+-supplied plants at aCO
2. Though eCO
2 tended to increase the ratio of total
15N content in the shoots vs. roots of plants treated with isotopes of both N forms at 33 °C, it significantly or non-significantly decreased the ratio of total
15N content in the shoots vs. roots in both
15NO
3− and
15NH
4+-supplied plants at 38 °C. As with plant dry mass and N-uptake rate, plants grown at eCO
2 plus chronic warming had the lowest ratio of total
15N content in the shoots vs. roots (
Figure 3). The ratio of total NO
3− in shoots vs. roots was significantly affected by both individual and interactive effects of CO
2 and temperature, while the ratio of NH
4+ in shoots vs. roots was affected only by CO
2 (
Figure S1). Elevated CO
2 significantly increased shoot:root NO
3− ratio at 33 °C, but not at 38 °C (
Figure S1A). Elevated CO
2 marginally decreased shoot:root NH
4+ ratio at both temperatures (
Figure S1B).
The ratio between organic N to total N was significantly affected by both CO
2 and the interaction of CO
2 × temperature (
Table S1). Elevated CO
2 tended to decrease the organic-N:total-N ratio at 33 °C (
Figure 4A). In contrast, the effect of chronic warming on the ratio between organic N to total N was dependent on the CO
2 treatment; chronic warming tended to increase the ratio at aCO
2, while it significantly decreased the ratio at eCO
2. Though the ratio between total
15N in plant proteins to total
15N in the plant of
15NO
3−-supplied plants was not significantly affected by CO
2 and/or temperature (
Table S1), it responded in a similar way as the ratio between organic N to total N in response to the independent variables (
Figure 4B). Furthermore, plants grown at eCO
2 plus chronic warming had the lowest ratio of
15N in total plant protein to
15N in the total plant. In addition, the ratio between total plant NO
3− to total plant N was greatest in the plants grown at eCO
2 plus chronic warming (
Figure 4C). Neither eCO
2 at 33 °C nor chronic warming at aCO
2 affected this ratio significantly, relative to aCO
2 and 33 °C. However, eCO
2 significantly increased the ratio between total plant NO
3− to total plant N at 38 °C.
Root %C was significantly affected by both temperature and CO
2, while root total nonstructural carbohydrate (TNC) was significantly affected by temperature and the interaction of temperature × CO
2 (
Table S1). Notably, both root %C and TNC tended to increase with the combination of eCO
2 plus chronic warming, compared with the other treatments (
Figure 5A,B). Root %N was significantly higher at 38 vs. 33 °C, while CO
2 had no significant effect on root %N (
Figure 5C,
Table S1).
In vitro activity (a measure of maximum potential activity) of NR in both leaves and roots was significantly increased at 38 °C vs. 33 °C. In addition, the in vitro activity of GS in roots also significantly increased at 38 °C. However, the in vitro activities of leaf GS and leaf and root GOGAT were unaffected by the higher temperature of 38 °C (
Figure 6).
3. Discussion
To date, most previous studies have focused on single-factor manipulation approaches when investigating the effects of global environmental changes (e.g., CO
2 enrichment, warming, drought, and N deposition) on plant N relations. However, as we expect these changes to occur concurrently in the future, multifactor manipulation approaches will be necessary to understand the impacts of climate change on plants. The combined effects of CO
2 enrichment and warming on plant N relations (uptake, translocation, and assimilation) were investigated in this study for this reason. As in our previous studies [
5,
48], the combination of eCO
2 plus chronic warming severely inhibited the growth of tomato, relative to either factor alone. Plants grown at eCO
2 plus chronic warming had the lowest N-uptake rate, net N translocation, and N assimilation when compared with plants grown at eCO
2 or chronic warming alone.
In general, it is expected that eCO
2 will increase plant biomass and supra-optimal temperatures (heat stress) will decrease plant biomass when water and nutrients are not limiting; this general trend was also observed in this study. However, the limited evidence from past studies shows that the interactive effects of eCO
2 plus warming on plant biomass can be highly variable. Some studies have shown eCO
2 plus warming to have a neutral or positive effect on plant growth [
49], while others have shown eCO
2 plus warming to have a negative effect [
5,
6,
7,
48]. Our growth reduction can be partly explained by decreased light interception of leaves, and, thus, in situ photosynthesis, caused by a vertical growth orientation of leaves (hyponasty) that occurs when tomato is grown under eCO
2 plus warming [
48]. The mechanism for eCO
2 + warming leaf hyponasty is not known, but, so far, appears to be restricted to compound-leaved species and is especially dramatic in tomato and potato. We have examined several tomato genotypes so far (hybrids and heirlooms, all indeterminant), and this hyponasty response occurs in all genotypes (with minor variation) [
48].
The effect of eCO
2 on plant N uptake has not been consistent [
12]. This is likely partly due to differences among studies in experimental protocols (e.g., length of CO
2 exposure, assaying intact vs. excised roots, differences in N source or amount) and partly due to naturally occurring variations among species [
12]. At least two possible mechanisms can explain how eCO
2 can affect N uptake: (1) in the short term, eCO
2 increases plant growth and, hence, plant N demand. This, in turn, increases N-uptake capacity [
12], and (2) eCO
2 induces stomatal closure and this decreases the transpiration-driven mass flow of N, which, in turn, decreases N uptake by roots [
9,
50]. However, in this study, eCO
2 did not affect either NO
3− or NH
4+-uptake rates at near-optimal growth temperature, but it did decrease uptake rates of both at 38 °C (and the effect eCO
2 + warming was non-additive). Further, plants grown at eCO
2 plus chronic warming had the lowest rates of NO
3− and NH
4+ uptake. Previously, we used sequential harvesting (vs.
15N labeling in this study) to show that the combination of eCO
2 (700 vs. 400 ppm) plus chronic warming (37 vs. 30 °C) can reduce root N-uptake rate of plants treated with either NO
3− or NH
4+ singly (not NH
4NO
3 as in this study) [
5]. This earlier study further showed that decreases in N-uptake rate may be due, at least in part, to decreased concentration or activity of N-uptake proteins (NRT1 and AMT1). Notably, chronic warming significantly increased NO
3−-uptake rate, but it did not influence NH
4+-uptake rate at aCO
2. An enhanced rate of NO
3− uptake with chronic warming could be due to the stimulation of uptake kinetics [
12], and the neutral effect of chronic warming on NH
4+-uptake rate could be to reduce excess accumulation of NH
4+ and avoid NH
4+ toxicity [
16]. Moreover, Bassirirad [
12] showed that the NH
4+:NO
3−-uptake ratio depends on soil or root temperature, and this ratio decreased as soil or root temperature increased from suboptimal to optimal levels (sufficient data were lacking to examine whether this statement holds true for optimal to supra-optimal temperature rises). Our results indicate this statement holds true even when temperature increases from optimal to supra-optimal levels. At 33 °C, NH
4+-uptake rate was approximately 1.4 times greater than NO
3−-uptake rate, regardless of the CO
2 treatment. However, when temperature increased from 33 °C to 38 °C, NO
3−-uptake rate increased to approximately 1.1–1.2 times that of NH
4+-uptake rate, regardless of the CO
2 treatment.
The ratio of total
15N in the shoots vs. the roots was used as a proxy for net N translocation from roots to shoots. Here, we were careful to use the term “net N translocation” instead of “N translocation”, as N can continuously circulate between roots and shoots. For example, foliar feeding of leaves with
15NO
3− has shown that leaf NO
3− can be translocated to every part of the plant, including the root system [
51]. Warm-season species, such as tomato, prefer shoot over root NO
3− assimilation, so most of the soil-derived NO
3− is translocated from roots to shoots and assimilated there [
52]. This is consistent with the high shoot-to-root
15N ratios (>4) for
15NO
3−-supplied plants in all four treatments in this study. Since NH
4+ assimilation generates H
+, and shoots have a limited capacity for proton disposal, nearly all soil-derived NH
4+ is assimilated in roots [
53]. Again, our results (low shoot-to-root
15N ratio of <2) are consistent with limited translocation of NH
4+ from roots to shoots in all four treatments. Although eCO
2 did not influence NO
3− or NH
4+-uptake rates, it tended to increase net N translocation of both
15NO
3− and
15NH
4+ in plants at 33 °C. This caused a significant increase in the ratio of total NO
3− in shoots-to-roots (
Figure S1A). The ratio of NH
4+ content in shoot-to-roots was significantly affected only by CO
2 (
p = 0.0285), and there was a trend for slightly lower shoot:root NH
4+ with eCO
2 regardless of the temperature (
Figure S1B). Though we did not measure photorespiration in this study, the slight decrease in shoot:root NH
4+ ratio with eCO
2 could be due to an inhibition of photorespiration by eCO
2. In C
3 plant leaves, NH
4+ flux from photorespiration is 5 to 10-fold higher than that from NO
3− reduction [
47]. Since limited soil-derived NH
4+ is typically translocated from roots to shoots, a higher percentage of shoot NH
4+ is likely represented by NH
4+ derived from photorespiration. Though chronic warming significantly increased NO
3−-uptake rate, it tended to decrease net N translocation in
15NO
3−-supplied plants at aCO
2. This resulted in a low shoot:root NO
3− ratio (
Figure S1A). As with NO
3− and NH
4+ uptake rate, the interactive effect of eCO
2 plus chronic warming caused a decrease in net N translocation from roots-to-shoots in both
15NO
3− and
15NH
4+-supplied plants. This also resulted in a low shoot:root NO
3− ratio in plants grown at eCO
2 plus warming (
Figure S1A).
The two ratios, total-plant organic N to total N, and total-plant
15N in proteins to total-plant
15N, were used as proxies for whole-plant N assimilation (decreases of these ratios denote inhibition), while the total NO
3−:total N ratio was used as a proxy for whole-plant NO
3− assimilation (increases of this ratio denote inhibition). Previous studies showed that eCO
2 can inhibit shoot NO
3− assimilation and enhance root NO
3− assimilation [
38,
41]. In this study, eCO
2 did not influence the ratio of
15N in proteins to total plant, but it decreased the organic N to total N ratio at 33 °C, indicating some inhibition of plant N assimilation. Moreover, total NO
3−:total N ratio was marginally increased with eCO
2 at 33 °C, which also indicates that eCO
2 may have a tendency to inhibit total-plant NO
3− assimilation. In contrast, chronic warming may slightly increase both organic N:total N and protein
15N:plant
15N at aCO
2, indicating a possible marginal stimulation of total-plant N assimilation. Chronic warming did not have an effect on total NO
3:total N ratio at aCO
2. However, these plants had the lowest total NH
4+:total N ratio (data not shown). Therefore, the slight stimulation of N assimilation by chronic warming at aCO
2 could have been due to the stimulation of NH
4+ rather than NO
3− assimilation. The combined effect of eCO
2 and chronic warming decreased both organic N:total N (significantly) and plant
15N:total
15N (marginally), indicating an inhibition of total-plant N assimilation. Moreover, eCO
2 plus chronic warming significantly increased total NO
3−:total N ratio, indicating an inhibition of NO
3− assimilation by eCO
2 plus chronic warming. Our results suggest the inhibition of whole-plant N assimilation by eCO
2 plus chronic warming was mainly due to inhibition of whole-plant NO
3− assimilation. However, based on root %N and total-root protein data, Jayawardena et al. [
5] suggested that eCO
2 plus chronic warming can inhibit root N assimilation in plants provided only NO
3− or only NH
4+. They further showed that, when plants were grown at eCO
2 plus warming, the roots had decreased levels of N-assimilatory proteins per gram root (i.e., nitrate reductase, NR, glutamine synthetase, GS, and glutamine oxoglutarate aminotransferase, GOGAT). Based on those results, we can assume that the inhibition of total-plant N assimilation by eCO
2 plus chronic warming could be due to decreased levels of these N assimilatory proteins. Further, we assessed the in vitro activities of assimilatory proteins extracted from plants grown at 33 °C, at both 33 and 38 °C. For all three proteins, the activities at 38 °C were greater or similar to 33 °C (
Figure S2), which confirms that these assimilatory proteins are not damaged by chronic warming temperature.
Previously, using wheat as the model species, Jauregui et al. [
4] reported that eCO
2 (700 vs. 400 ppm) plus warming (ambient + 4 °C) reduced leaf N assimilation by reducing energy availability. Since plants grown at eCO
2 plus chronic warming had the lowest N-uptake rates and N assimilation, we hypothesized that this could be due to the lower energy or resource availability in roots to perform root functions, which we tested by measuring root %C, TNC, and %N. Relative to the other treatments; plants grown at eCO
2 plus warming had the highest root %C and TNC concentrations, while they also had the co-highest root %N. Based on these results, we concluded that the low rates of N uptake and N assimilation in plants grown at eCO
2 plus warming were not due to limited energy or resource availability in roots.
In summary, tomato plants grown at eCO
2 plus chronic warming had the lowest plant dry mass, NO
3− and NH
4+-uptake rates, net N translocation, and whole-plant N (and NO
3−) assimilation when compared with other treatments. Previously, we showed that N uptake can be inhibited by eCO
2 (700 vs. 400 ppm) plus warming (37 vs. 30 °C), using a sequential harvesting technique [
5]; furthermore, in this study, we showed that both NO
3− and NH
4+-uptake rates can be inhibited by eCO
2 plus warming using
15N labeling. Moreover, in this study, we showed that net N translocation can be inhibited by eCO
2 plus warming using two different methods: (1) the
15N ratio between shoots and roots, and (2) the NO
3− ratio between shoots and roots. Finally, we showed that the whole-plant N assimilation can be inhibited by eCO
2 plus warming using two methods: (1) the ratio between organic N and total N, and (2) the ratio between
15N in proteins and
15N in the plant). Inhibition of whole-plant N assimilation was mainly due to the inhibition of NO
3− assimilation by eCO
2 plus chronic warming. In addition, the decreased rates of N uptake and N assimilation were not due to the resource limitation (N or C) for root functions, but, probably, due to the decreased levels of enzymes involved in N metabolism (NR, GS, GOGAT), as shown previously [
5]. Overall, this study has shown that the interactive effects of CO
2 enrichment and global warming can negatively affect plant N metabolism in tomato, which will have serious consequences for the production and nutrient quality of tomato, one of the world’s most-important non-grain food crops. Given global human population is expected increase by 1.4 to 3.9 billion by 2050, global crop production will need to increase by then by ca. 70% to meet global food demand [
54]. This study provides valuable information regarding weak links in N metabolism in response to CO
2 enrichment and global warming that can be targeted for improvement, in order to improve yield and nutrient quality of tomato, and, perhaps, other crops in the future.