Continued increases in atmospheric carbon dioxide (CO₂) and other greenhouse gases will continue to cause long-lasting adverse effects on global climate, such as rising global temperatures and altered precipitation patterns [1] . These climate changes can also influence environmental factors, including soil nitrogen availability, ultimately altering plant and ecosystem productivity [2] [3] . The burning of fossil fuels has substantially increased atmospheric nitrogen deposition into soil over the last century and the increase is expected to continue in the foreseeable future [4] - [6] . Furthermore, climate change can modify soil nitrogen availability by affecting litterfall and litter decomposition rates in forests [2] .

Elevated [CO₂] generally increases photosynthesis and plant growth, but these responses depend on site conditions, such as soil nitrogen (N) availability [7] - [10] . Soil nitrogen is a critical component of photosynthetic enzymes, making soil nitrogen availability a key regulator of plant responses to elevated [CO₂] [11] . Elevated [CO₂] can affect soil nitrogen concentration by influencing plant growth and nitrogen uptake. When plants grow vigorously, their demand for nitrogen increases [12] [13] . Moreover, elevated [CO₂] can also affect soil nitrogen by influencing the amount and quality of litter and its decomposition [14] - [17] .

Photosynthesis is a primary physiological process through which nitrogen supply affects plants [8] [18] [19] . Elevated [CO₂] enhances photosynthesis by increasing Rubisco carboxylation rates and substrate supply, while suppressing Rubisco oxygenation [20] - [23] . However, elevated [CO₂] can cause the downregulation of photosynthetic machinery as demonstrated in both FACE and controlled-environment experiments [24] [25] . Photosynthetic downregulation has been reported for many plant species grown under limited nitrogen availability and elevated [CO₂] [26] - [31] . This downregulation is often linked to tissue nitrogen dilution caused by excessive carbohydrate accumulation under elevated [CO₂], which reduces the mass based leaf nitrogen concentration [32] - [34] . Also, the excessive accumulation of nonstructural carbohydrates suppresses the expression of genes for Rubisco synthesis [35] [36] . However, some studies have found no correlation between photosynthetic downregulation and leaf nitrogen concentration or nitrogen supply, highlighting the complexity of these interactions [37] [38] . Other studies have found that plants increase nitrogen allocation to roots at the expense of above-ground organs under elevated [CO₂] [39] - [42] . Thus, a good understanding of how nitrogen supply affects the physiological and morphological responses of plants to elevated [CO₂] is critical for predicting the performance of plants and plant communities under future climate conditions [43] . Moreover, elevated [CO₂] can reduce nitrate assimilation in the leaf by reducing photorespiration, which in turn will increase plant nutrient demand and may result in the eventual depletion of the nutrient pool in the soil. If the plant is not efficiently assimilating nitrate, it might compensate by increasing its nutrient uptake from the soil, creating a higher demand, which will result in the depletion of nutrient pools in the soil over time, affecting the overall nutrient balance [44] - [47] .

Yellow birch (Betula alleghaniensis Britt.) is a deciduous tree species native to North America with a medium growth rate [48] . It is highly valued both ecologically and commercially. As a hardwood species, it is prized for its heavy, strong, and close-grained wood, making it a significant source of lumber for quality products such as furniture, cabinetry, interior finishes, veneers, tool handles, boxes, woodenware, interior doors, charcoal, and pulp [49] [50] . Additionally, it serves as an important food source for both livestock and wildlife [50] . Beyond its commercial uses, yellow birch also contains bioactive compounds with promising applications in the food, pharmaceutical, and cosmetic industries [51] . Its ecological importance is equally noteworthy. As an early colonizer, yellow birch has been observed to play a key role in rehabilitating disturbed areas, such as sites damaged by brine from well-injection fluids. Its ability to establish itself as soil salinity levels return to near-normal demonstrates its potential for ecosystem recovery [52] . However, yellow birch is highly sensitive to environmental and climatic changes, facing significant challenges such as high mortality rates under drought and freeze-thaw conditions [53] [54] . Its shallow root system further limits adaptability, contributing to its vulnerability in degraded forest stands and under global environmental changes [55] . While yellow birch demonstrates physiological and morphological responses to environmental shifts, studies on these responses are inconsistent or contradictory. For example, Song and Cheng [53] report that elevated CO₂ increases biomass allocation to roots, while Wang et al. [31] find elevated CO₂ reduces biomass allocation to roots. Its responses to CO₂ are affected by other environmental factors, such as soil moisture [56] . While there are numerous studies investigating the effect of nitrogen supply on the responses of other birch species to elevated CO₂ in recent years, For instance, elevated CO₂ and increasing nitrogen supply synergistically enhance the photosynthesis and biomass production of white birch [57] - [60] . Wang et al. [30] report that a higher nitrogen supply reduces the scope of photosynthetic downregulation induced by elevated CO₂ and warmer temperatures in B. costata. However, there is no such information in the literature on Betula alleghaniensis. In this study, we investigated the interactive effects of CO₂ and nitrogen supply on the physiological and morphological performance of Betula alleghaniensis. Since our experimental protocols are similar to those of [31] , we hypothesized that the responses of Betula alleghaniensis to CO₂ and nitrogen supply would be similar to those of B. costata, i.e., elevated CO₂ would lead to photosynthetic downregulation and the scope of the downregulation would be smaller under higher nitrogen supply, and elevated CO₂ and higher nitrogen supply would both tree growth and biomass production but reduce biomass allocation to roots.

The elevated [CO₂] increased seedling root collar diameter growth and shoot biomass, and the increases were greater with higher nitrogen supplies. These synergistic effects were also shown seedling height growth (150 mg·N·L⁻¹ nitrogen vs. lower levels). These results support our hypothesis that the stimulation of growth by elevated [CO₂] would be higher with increasing N supply. The results are in general agreement with [58] on white birch (B. papyrifera Marsh.) and [31] on yellow birch B. costata. The synergistic effects may be attributable to the increases in nitrogen use efficiency by elevated [CO₂] [65] - [67] . Increases in aboveground growth by elevated [CO₂] are common in the literature [68] [69] . However, while the lack of significant positive effect of elevated [CO₂] on height growth in this study is consistent with the findings for some other tree species such as Eucalyptus miniata [70] and Populus [71] , explanations are needed. Height growth is a key trait determining the competitiveness of plants in aboveground competition [72] [73] and has a higher priority in carbohydrates allocation than diameter growth and defense and stress resistance [74] . Therefore, if the need of carbohydrates for height growth is satisfied under the ambient [CO₂], [CO₂] elevations will not future increase height growth.

Growth is generally positively correlated with specific leaf area (SLA) in trees [75] - [78] . However, the elevated [CO₂] treatment in this study significantly reduced specific leaf area (SLA) of yellow birch in all the nitrogen treatments. Similar results have been reported for white birch [58] , barley and tomato plants [79] . The reduction in specific leaf area (SLA) by elevated [CO₂] was most likely the result of increased carbohydrates accumulation rather than changes in leaf structure or anatomy [80] [81] .

Both elevated [CO₂] and increasing nitrogen (N) supply increased the stem dry mass, leaf dry mass and total dry mass of yellow birch. These results are consistent with the findings of several other studies [31] [56] [58] [82] [83] . Although plants generally downregulate their photosynthetic capacity in response to elevated [CO₂], the actual rates of photosynthesis generally increase, leading to an increase in biomass production and growth [84] . Increases in photosynthesis and biomass production under increased nitrogen supply are generally related to increases in nitrogen allocation to photosynthetic apparatus and subsequent increases in carbohydrate production [85] - [87] . The synergistic effects of elevated [CO₂] and higher nitrogen supply, as found in this and other studies (e.g., [88] [89] ), are attributable to the increase of nitrogen use efficiency under elevated [CO₂] [65] - [67] . Effects of elevated [CO₂] on root biomass, however, are highly variable: from no effects in this study to increases in [90] . These contrasting responses of roots to elevated [CO₂] appear to be related to genetic differences [90] . Nonetheless, our results indicate that yellow birch growing on rich sites will likely benefit more from the continued elevation of [CO₂] than their counterparts on poor sites.

The response of biomass allocation to elevated [CO₂] varies among different studies. Stem mass ratio (SMR) increased in response to elevated [CO₂] in this and some other studies [91] [92] . [88] report elevated [CO₂] stimulates the stem diameter growth of Larix sibirica. [81] suggest that elevated [CO₂] increases stem biomass density. Some older studies, in contrast, find not significant [CO₂] effect on biomass allocation between roots and shoots [93] - [96] . The differences in the response may be attributable to differences in other environmental factors, such as nitrogen supply. This and other studies [97] - [99] find that [CO₂] and nitrogen supply interactively affect biomass allocations in trees. The results that elevated [CO₂] reduced biomass allocation to roots and the reduction was greater under higher nitrogen supply are consistent with the findings of [31] [100] . The results are also in agreement with the results of long-term FACE studies that elevated [CO₂] has greater positive effects on aboveground growth than roots under higher nitrogen supply [24] [89] [101] . The consistent findings of short-term controlled environment studies and long-term field research indicate that trees growing on nutrient-rich sites will benefit more from elevated [CO₂] than those growing on poor sites and that fertilization in the future under elevated [CO₂] will be more effective in increasing forest productivity. Additionally, elevated [CO₂] can reduce nitrate assimilation in leaves by suppressing photorespiration, a process critical for converting nitrate into organic forms within the plant. This reduction in nitrate assimilation, combined with increased growth and biomass production stimulated by elevated [CO₂], significantly raises overall nutrient demand, particularly for nitrogen. The increased nutrient demand may lead to compensatory uptake of soil nutrients, potentially depleting soil nutrient pools over time, which in turn can alter the biomass allocation between aboveground and below-ground organs due to variations in soil nitrogen availability [102] . While these results indicate that the performance of seedlings can serve as a reliable indicator of tree’s response [103] , this and other controlled environment studies do have limitations, for example, they do not capture the full complexity of natural ecosystems or the fluctuations of environmental conditions at nature sites. Therefore, cautions should be exercised when applying the results from controlled environment studies to the field or even computer modeling.

It is interesting to note that elevated [CO₂] increased photosynthesis, but N supply had no significant effect on photosynthetic rates in yellow birch in this study. In response to the elevation of [CO₂], the photosynthetic rate of yellow birch was more than doubled. However, the photosynthetic rates were relatively stable among different levels of nitrogen supply. In contrast, the total leaf biomass per tree increased linearly with increases in nitrogen supply. These results suggest that the primary mechanism of response to variation in nitrogen supply in yellow birch was morphological rather than physiological. This conclusion is consistent with that [30] . Our findings suggest that it is critically important to assess different mechanisms of responses in order to obtain the whole picture of the tree’s response. Furthermore, it is interesting to note there are large variations in measured photosynthetic rates of B. Allegheniensis among different studies. For example, [30] report 20 µmol·m⁻²·s⁻¹ under elevated [CO₂], it varies from 7 to 12 µmol·m⁻²·s⁻¹ under elevated [CO₂] and from about 7 to 9 µmol·m⁻²·s⁻¹ under ambient [CO₂] in [56] while the rate ranged from about 5 µmol·m⁻²·s⁻¹ under ambient [CO₂] to about 11 µmol m⁻²·s⁻¹under elevated [CO₂] in this study. The large differences appear to be related to seed source, temperature and soil moisture. Although the three studies use the same species, the seeds are from very different geographic locations. The growth temperatures in [30] [56] are much higher than the values in this study. [30] used 25/16˚C day/night temperature for ambient [CO₂] treatment and 29/20˚C for elevated [CO₂] treatment while [56] used 26/19˚C day/night temperature for both ambient and elevated [CO₂]. We emulated the day and seasonal (June 4^th to September 30^th) temperatures of the seed source site throughout the entire experiment and the actual temperatures (21/13˚C day/night average) were several degrees lower than the other two studies and varied with time of day and from week to week. The temperature that we used to measure photosynthesis was also much lower than the other two studies. Besides the direct temperature effects, soil moisture could also have an indirect effect. Under similar irrigation schemes, soil moisture should be lower at higher temperatures because of increases in evaporation from the pots and higher rates of water loss from transpiration. [56] find the photosynthesis of B. Allegheniensis increases with decreases in soil moisture. These results indicate that it is critically important to consider the growth and measurement conditions when quoting or using published values of photosynthesis and other physiological parameters in the literature.

[CO₂] and nitrogen supply interactively affected J_max but not V_cmax. Elevated [CO₂] increased both photosynthetic rate and V_cmax and the increases were not influenced by nitrogen supply. As discussed previously, the lack of interaction between [CO₂] and nitrogen levels was related to the primary response mechanism of yellow birch to nitrogen supply. However, the direction of J_max response to nitrogen supply was opposite under different [CO₂] treatments. These results are in contrast to the findings of [104] on black spruce where [CO₂] and nitrogen availability interactively affected V_cmax but not J_max. The different responses of V_cmax and J_max may reflect a shift in primary limitation to photosynthesis between Rubisco limitation and RuBP regeneration limitation [30] . However, similar studies in yellow birch have reported photosynthetic downregulation under elevated [CO₂] and warmer temperatures, attributing it to a significant dilution of leaf nitrogen concentration, as evidenced by a substantial increase in total leaf area [30] [31] . Photosynthetic downregulation typically occurs when growth enhancement by a specific factor results in a dilution of leaf nitrogen concentration. Increasing nitrogen supply can reduce or avoid the dilution of leaf nitrogen and photosynthetic downregulation [27] [30] .

It is interesting to note that elevated [CO₂] had no effect on stomatal conductance and transpiration. In general, elevated [CO₂] leads to decreased stomatal conductance and transpiration [105] - [108] . In the context of climate change, elevated [CO₂] reduces stomatal conductance while increasing water use efficiency (WUE), which is beneficial for plant development, especially when drought conditions are also projected to worsen [109] [110] . While the reasons for the contradicting results are not entirely clear, the lack of stomatal response to elevated [CO₂] is not uncommon in the literature [111] - [114] . There are even studies reporting increased stomatal conductance (g_s) in response to elevated [CO₂] in some plant species [110] - [116] . Thus, it can be concluded that the negative effect of elevated [CO₂] on stomatal conductance is a general rather than universal response [117] .

In summary, the results of this study suggest that yellow birch may increase their photosynthetic capacity, biomass production, and growth in the future when both [CO₂] will be higher, particularly on nutrient-rich sites, due to continued increases in [CO₂] emissions. Although increasing nitrogen deposition due to continued climate change may also have positive effects on tree’s response to elevated [CO₂], nitrogen deposition can have significant effects on the nutrient balance in soil, potentially influencing the availability and cycling of other nutrient elements. For instance, excessive nitrogen deposition without a proportional increase in phosphorus can lead to a nitrogen-phosphorus imbalance, altering soil microbial communities and their activities and also may increase the leaching of mobile nutrients, including potassium and calcium, which eventually lead to nutrient loss from the soil, potentially impacting the nutrient balance and fertility of the soil [118] - [121] . However, the biomass allocation between aboveground and belowground organs may be altered due to the variation in soil nitrogen availability. Additionally, the results of this study further underline the importance of considering the interactive effects of [CO₂] and other environmental factors, such as soil nitrogen availability, for predicting the growth, survival and productivity of plants and plant communities under the future climate conditions.

The findings from this study highlight significant implications for forest management under future climate change scenarios. The positive response of yellow birch to elevated [CO₂] levels, particularly under sufficient nitrogen availability, suggests opportunities to optimize forest productivity through targeted nutrient management strategies. Specifically, nitrogen fertilization in nutrient-poor sites could enhance yellow birch growth, improving both timber yields and carbon sequestration potential. Furthermore, the increased growth of yellow birch under these conditions indicates its suitability for reforestation and ecological restoration projects, making it a valuable species for mitigating climate change impacts. These results align with or diverge from findings in other tree species, providing a broader context. For instance, the reduction in root biomass allocation under elevated [CO₂] mirrors patterns seen in Betula costata and B. papyrifera, yet contrasts with species such as Larix sibirica, where root allocation remains less affected. These variations suggest that species-specific traits and environmental interactions play a critical role in shaping biomass allocation patterns under elevated [CO₂]. Additionally, while photosynthetic downregulation is commonly observed under elevated [CO₂] and limited nitrogen availability in many tree species, such as Pinus taeda and Quercus robur, yellow birch demonstrated stable photosynthesis across nitrogen levels in this study. This stability may reflect unique resilience mechanisms in yellow birch, providing insights for selecting climate-resilient species for forest management. However, the results also underline potential challenges related to soil nutrient depletion due to increased nutrient demand under elevated [CO₂] conditions. Forest managers must implement sustainable practices, such as balanced fertilization and monitoring of nutrient cycling, to maintain soil health and ensure the long-term productivity of forest ecosystems.

Finally, the synergistic effects of elevated [CO₂] and nitrogen supply on biomass production highlight the importance of nutrient availability for maximizing forest productivity. These results align with findings from long-term FACE experiments, suggesting that forests on nutrient-rich sites are likely to benefit more under future elevated [CO₂] conditions. However, the interactive effects observed in this study underscore the need to incorporate species-specific responses and environmental variability into predictive models of forest dynamics under climate change.

We emulated the seasonal photoperiod and daily and seasonal temperature conditions of the seed sources in our experiment and the results are in general agreement with those of longer-term FACE studies in the field. Therefore, the results may be more applicable to trees growing in the field than those from controlled environment studies where the environmental conditions are held constantly through the experiment. However, our study still has limitations. First, there were only two replicates for the [CO₂] treatment (2) due to the limitation of our greenhouse infrastructure. Second, the treatments were carried out on tree seedlings. The physiological response of trees to treatments can vary with the size and developmental stage. Third, the hourly temperatures that we used were average values of the site for the past 10 years and the actual conditions in the field fluctuate from day to day and year to year. Therefore, the experiment did not capture the effects of fluctuations in temperature, precipitation, nutrients and other environmental factors in the field. Fourth, we grew the trees in pots, which limit the extension and development of roots. Therefore, the results of this study should be used with caution and interpreted in the proper context. Additionally, the relatively short duration of the experiment (four months) limits our ability to assess the sustained impacts of elevated [CO₂] and nitrogen supply on yellow birch. Longer-term studies are needed to determine whether the observed responses are consistent over time or if they change as trees mature and as soil nutrient pools are depleted or altered. Furthermore, the controlled environment conditions do not include interactions with soil microbiota, herbivory, and other biotic factors that could significantly influence tree responses in natural ecosystems. Future studies should address these limitations by incorporating longer experimental durations, conducting field-based research, and including mature trees to better understand the long-term and ecosystem-level impacts of elevated [CO₂] and nitrogen supply on yellow birch and other forest species.