Stem N 2 O flux seasonality relates to physiological activity

All boreal trees studied showed substantial seasonality in their N 2 O exchange. Previous rare studies have focused only on short periods of the vegetation season, excluding measurements in the dormant winter season. High and constant stem N 2 O emissions were observed from all the tree species studied during spring and summer months (April–September), independently of soil VWC. This was followed by a decrease from October onwards (Fig. 1). Tree fluxes remained low in winter and increased again in March. Stem CO 2 effluxes revealed similar seasonality as did N 2 O fluxes (Supplementary Fig. 1). Seasonal dynamics of accompanying environmental parameters are presented in Fig. 2. A strong positive relationship between stem N 2 O and CO 2 fluxes was detected (ρ = 0.714–0.745, depending on tree species; p < 0.001; Spearman’s rank correlation), and reduced flux rates were observed in the dormant season (Fig. 3). The positive relationship was further supported by a partial least squares (PLS) path analysis (Fig. 4, Supplementary Figs. 2, 3). Based on the results of the PLS path and correlation analyses, the second main driver of the stem N 2 O fluxes is the GPP of the forest (ρ = 0.543–0.660, p < 0.001; Fig. 4, Supplementary Figs. 2, 3). For birch and pine, the stem CO 2 efflux and GPP together explained 44% and 37%, respectively, of the variance in the stem N 2 O fluxes (Supplementary Figs. 2, 3).

Fig. 1 Stem and forest floor N 2 O fluxes. Seasonal courses of monthly N 2 O fluxes (mg m−2 month−1) and total annual N 2 O fluxes (mg m−2 yr−1) from stems of birch (a), spruce (b), and pine (c), and from forest floor (d) measured from June 2014 to May 2015. Positive fluxes indicate N 2 O emission, negative fluxes N 2 O uptake. The solid line within each box marks the median value, broken line the mean, box boundaries the 25th and 75th percentiles, and whiskers the 10th and 90th percentiles. Statistically significant differences among annual fluxes at p < 0.05 are indicated by different letters above bars. Mean annual volumetric water contents (± standard error) of the plots were as follow: wet plot, 0.81 ± 0.02 m3 m−3; moderately wet plot, 0.40 ± 0.02 m3 m−3; and dry plot, 0.21 ± 0.01 m3 m−3. The dry plot did not have spruce or birch trees. Stem fluxes were measured from three trees per species at each plot (n = 3). Forest floor fluxes were measured at three positions at the wet and moderately wet plots (n = 3) and at six positions at the dry plot (n = 6). Annual fluxes were calculated as the sums of 12 monthly fluxes Full size image

Fig. 2 Seasonal courses of basic environmental variables. The variables were measured at the SMEAR II station from June 2014 to May 2015: (a) Daily mean air temperature within the forest stand at 8 m height; (b) daily mean soil temperature and (c) soil volumetric water content (VWC), both in C1-horizon (38–60 cm in depth); and (d) daily sum of photosynthetically active radiation (PAR). The open circles represent monthly means (± standard error), and the black circles indicate monthly means ( ± standard error) calculated for the flux measurement days only. The period of continuous snow cover (from mid-December 2014 to early April 2015) is indicated in Fig. 2b Full size image

Fig. 3 Relations between N 2 O and CO 2 stem fluxes. N 2 O versus CO 2 fluxes in stems of birch (a), spruce (b), and pine (c) measured from June 2014 to May 2015. Data for dormant (October–February) and vegetation (March–September) seasons are indicated. The stem fluxes were measured from three trees per species at three studied plots characterised by mean annual volumetric water content as wet (0.81 ± 0.02 m3 m−3; mean ± standard error), moderately wet (0.40 ± 0.02 m3 m−3), and dry (0.21 ± 0.01 m3 m−3). The dry plot did not have spruce or birch trees. All fluxes are expressed per m2 of stem surface area. Positive flux values indicate gas emission, negative values gas uptake Full size image

Fig. 4 Prediction of N 2 O fluxes in spruce stem. Path diagram, created on the base of partial least squares path modelling, describes relationships among stem N 2 O fluxes and most predictive environmental, physiological, and ecosystem variables (drivers of N 2 O fluxes) for 2014–2015. Values in circles report coefficients of determination (R2). Values included in arrows mark the path coefficients, whose significance levels are expressed as follows: *p < 0.05, **p < 0.01, ***p < 0.001. Soil volumetric water content (Soil VWC), photosynthetically active radiation (PAR), gross primary production (GPP). Soil N 2 O flux expresses forest floor N 2 O flux Full size image

From the available environmental variables, the stem N 2 O fluxes of all the studied tree species correlated positively (p < 0.001) with air temperature (ρ = 0.559–0.645), as well as intensities of photosynthetically active radiation (PAR) (ρ = 0.513–0.637) and ultraviolet (UV) radiation (ρ = 0.541–0.694). We also found strong positive correlation between the stem N 2 O fluxes and ecosystem evapotranspiration (ρ = 0.536–0.688, p < 0.001). That relationship was not proven by the PLS path analysis, however.

The relatively strong relationship between the stem N 2 O fluxes and those variables reflecting the physiological activity of the trees and ecosystem as a whole (stem CO 2 efflux, GPP, evapotranspiration) suggests that the stem N 2 O fluxes do not constitute merely a passive process based on N 2 O concentration gradients in the soil–stem–atmosphere continuum. The possible coupling between N 2 O and CO 2 fluxes was early detected in species belonging to cryptogamic covers11,24 and Spermatophyta11,25. This assumption is based on the finding of constant N 2 O:CO 2 emission ratio under a wide range of controlled environmental conditions24,25. However, here we show for the first time a tight linear correlation between N 2 O and CO 2 fluxes even in adult trees during the whole vegetation period (Fig. 3) supporting thus the hypothesis of physiologically dependent N 2 O exchange in tree stems.

The strong positive correlation with evapotranspiration supports our hypothesis that N 2 O is taken up from the soil by roots, then transported into the above-ground tree tissues in xylem via the transpiration stream. This hypothesis is supported also by the good solubility of N 2 O in water26 and the demonstrated ability of plants lacking the aerenchyma system8,9,13 to transport N 2 O from the soil and emit it through the stem. To our knowledge, this is the first study showing that N 2 O exchange by mature boreal tree stems is closely connected to the physiological activity of trees and ecosystem, particularly to processes of carbon release and uptake, including stem CO 2 efflux and GPP. Future experimental studies are needed, however, to confirm that transpiration rate drives N 2 O emissions from tree stems.

Forest floor N 2 O flux together with the stem CO 2 efflux and GPP explained 45% of the stem N 2 O flux variance in spruce (Fig. 4). Spruce was the only tree species manifesting a weak relationship between stem and forest floor N 2 O fluxes (ρ = 0.353, p < 0.001) (Fig. 4). Similarly, a positive but weak correlation between stem and forest floor N 2 O fluxes was found earlier in pine trees grown in the same forest13. Lack of strong correlations indicates a partial decoupling of stem and forest floor N 2 O fluxes. Generally, net fluxes at the tree stem/soil–atmosphere interface reflect a balance between processes of production, consumption, and transport of N 2 O within trees and soil from the sites of production to the sites of release27. Accordingly, substantial variation in root depth among tree species can contribute to observed species specificity in N 2 O fluxes. The net forest floor N 2 O flux does not necessarily reflect the N 2 O concentration or production/consumption in the rooting zone11, because the plants can directly alter soil microbiological N turnover processes28,29,30,31 by modifying the quantity and quality of soil organic matter, nutrient availability, and soil pH29,32. This includes release of exudates33 and radial oxygen loss34 from the roots, which are again closely connected to such tree physiological processes as photosynthesis35. Furthermore, leaf and root litter quality and soil water uptake by trees, both of which are specific to tree species, can substantially affect the N cycling processes in soils28. Production of N 2 O in soil is further directly and indirectly influenced by an activity of mycorrhizal fungi via the modulation of denitrification processes and physio-chemical soil properties regulating N 2 O turnover like carbon, nitrogen, and water availability, as well as soil aeration and promotion of soil aggregation, respectively36,37,38,39. Mycorrhizal fungi itself seem to possess also the ability for denitrification and might be therefore important sources of N 2 O36,38. Due to these strong rhizospheric effects of plants and mycorrhizal fungi on soil N turnover processes, the ratio between N 2 O production and consumption in the soil might also be highly variable. Hence, the availability of N 2 O in the rhizosphere affects the N 2 O uptake by tree roots and subsequent N 2 O emissions from tree surfaces into the atmosphere, and that might not be reflected in the overall net N 2 O exchange at the soil surface.

In addition to that of soil origin, N 2 O emitted by trees can also be formed directly in the tree tissues. The direct N 2 O production in plants is proposed to originate from microorganisms living in association with the plants36, as described earlier with mycorrhizal associations, or from N 2 O produced via photo-assimilation of NO 3 − in photosynthetically active tree tissues10,21,22, or via a newly detected biotic pathway with mechanisms different from known microbial or chemical processes25, or via an abiotic UV-dependent process on leaf surfaces40. The plant’s own N 2 O production process seems to be light dependent, requiring energy from primary photosynthetic reactions10,12,23. The mechanisms and processes behind radiation induced N 2 O emissions are still poorly understood, however, and especially with respect to mature trees. Moreover, the possible contributions of the various N 2 O production processes in plants to the net N 2 O fluxes at the tree–atmosphere continuum are largely unknown. To the best of our knowledge, only Machacova et al.13 have reported N 2 O emissions from leaves of mature trees and showed that the leaf emissions might considerably exceed the emissions from the stems and could therefore constitute an additional source of N 2 O in forest ecosystems13.

In conclusion, the N 2 O emission rates from tree stems show clear seasonal dynamics with the highest emissions detected during summer months when also air temperature, PAR and UV intensities are the highest. The seasonal changes in N 2 O emission closely relate to the physiological activity of trees associated with CO 2 exchange as demonstrated by a tight linear correlation between N 2 O and CO 2 fluxes.

Boreal trees exchange N 2 O even during dormant season

Based on the seasonal changes in stem CO 2 efflux (Supplementary Fig. 1a–c), the period from October to February was identified as a dormant season. In addition to the vegetation-season N 2 O emissions, our study revealed that all the studied tree species can emit N 2 O even during the dormant season, and particularly on the plot characterised by high soil VWC (i.e. the wet plot). At this plot, the stem N 2 O emissions over the dormant season contributed from 2% (birch) to as much as 16% (spruce) to the annual N 2 O emissions (Fig. 5a‒c).

Fig. 5 Seasonal N 2 O fluxes in tree stems and forest floor. N 2 O fluxes in stems of birch (a), spruce (b), and pine (c), and in forest floor (d) are presented at annual scale (black columns), for vegetation season (March–September, grey columns), and for dormant season (October–February, white columns). The fluxes (means ± standard error) are sums of N 2 O exchanged over one year, vegetation season, or dormant season, respectively, and expressed per m2 of stem or soil surface area. Positive flux values indicate N 2 O emission, negative values N 2 O uptake. Mean annual volumetric water contents ( ± standard error) of the plots were as follow: wet plot, 0.81 ± 0.02 m3 m−3; moderately wet plot, 0.40 ± 0.02 m3 m−3; and dry plot, 0.21 ± 0.01 m3 m−3. The dry plot did not have spruce or birch trees. Stem fluxes were measured from three trees per species at each plot (n = 3). Forest floor fluxes were measured at three positions at the wet and moderately wet plots (n = 3) and at six positions at the dry plot (n = 6). Statistically significant differences between fluxes over vegetation and dormant season at p < 0.05 are indicated by asterisks. The percentage contributions of fluxes over the vegetation and dormant season to the annual fluxes (defined as 100%) are indicated above the bars Full size image

The small but detectable winter N 2 O fluxes of the tree stems were accompanied by low but consistent CO 2 emissions from the stems (Supplementary Fig. 4a‒c), thereby reflecting the rate of maintenance respiration during the dormant period41. This is supported by the fact that air temperatures were generally mild on the measurement days (Fig. 2a). It has been shown that stem CO 2 effluxes in boreal trees decrease significantly during winter periods, when stems are frozen42. Large amounts of CO 2 can nevertheless be released in short-term CO 2 burst events during freezing and thawing of tree stems and thus contribute significantly to the seasonal CO 2 dynamics42. We did not observe such bursts during autumn measuring campaigns when the air temperature was above zero, but slightly elevated stem CO 2 fluxes during February (pine) and March (birch, spruce) might indicate CO 2 bursts from freezing and thawing tree stems (Supplementary Fig. 1). The stem N 2 O flux dynamics, albeit at comparatively lower rates, follow a similar seasonality in spring (Fig. 1), thus supporting the idea that both CO 2 and N 2 O originate from a similar source. Both gases dissolved in the xylem sap might be released from the stem during freezing to avoid winter embolism in the xylem conduit and during thawing from the intercellular spaces, where gases can be trapped during the process of stem freezing42.

On plots characterised by lower soil VWC, the stems even consumed N 2 O from the atmosphere during the dormant season, thus contributing to reduction of the annual source strength of trees and the ecosystem as a whole (Fig. 5a–c). Birch was identified as the strongest N 2 O sink. Dormant uptake by birch stems amounted to as much as 52% of the annual N 2 O emissions at the moderately wet plot (Fig. 5a). We speculate that the species variability in N 2 O exchange (Figs. 1, 5) might be explained by spatial variability of N 2 O concentration in soil, which is more pronounced under lower soil VWC. Under such conditions, N 2 O sources are more diverse due to simultaneously running aerobic and anaerobic N turnover processes leading to production and consumption of N 2 O. Under dry conditions, therefore, root depth and distribution seem to play a more important role, species specificity is more pronounced, and differences among individual trees having different N 2 O sources available also are more prominent. This hypothesis should be confirmed by further research.

To the best of our knowledge, the limited number of studies reporting N 2 O exchange of tree stems present trees only as N 2 O emitters13,14. The only tree species known able consistently to take up N 2 O from the atmosphere is European beech11. That species’ cryptogamic stem covers were shown to be organisms that might be co-responsible for beech’s uptake of N 2 O. In their study, Machacova et al. (2017)11 observed that N 2 O consumption rates were closely related to the respiratory CO 2 fluxes of trees and cryptogams, thus indicating a connection between N 2 O consumption and the physiological activity of trees and microbial communities. Our observed N 2 O consumption by boreal tree stems is probably not linked to the physiological activity of the cryptogams associated with the tree bark, because during the dormant season any physiological activity in the forest is very low, as evidenced by the negligible stem CO 2 effluxes. We hypothesise that the reason for high N 2 O uptake observed in birch trees might be that birch trees, in contrast to the studied conifers, possess an aerenchyma system serving as a passive gas transport pathway within the tree43. Under low winter N 2 O concentration in soil, the broadleaf birch trees might hypothetically take up N 2 O from the atmosphere through lenticels in the bark, transport this gas along the concentration gradient into the roots, then perhaps release it into the soil at the root tips lacking exodermis. In contrast to the wood of conifers, that of the birches is a diffuse-porous type44 that is more gas-permeable45. The N 2 O might also be reduced by denitrifiers directly in stem tissues below the lenticels, although such microbial activity would be decreased in wintertime6,46. At least in the case of Betula potaninii, it seems also that the vapour phase-based water and oxygen permeance of individual lenticels is significantly reduced during wintertime due to the production by phellogen of compact tissues closing off lenticels at the end of the vegetation season. These tissues lacking intercellular spaces reduce gas exchange between the atmosphere and the system of intercellular spaces within the stem47. Hence, the mechanisms behind the uptake of N 2 O by trees and the fate of N 2 O remain unknown.

Similarly to the tree stem fluxes, the forest floor was a source of N 2 O in the vegetation season independently of the soil VWC (Fig. 5d). The rates of N 2 O emission were in line with those reported earlier for the same forest48. The similarly elevated emissions at all the studied plots in September (Fig. 1d) might be connected to litterfall, which is regarded as the largest external N input to the soil and hence suggested to stimulate N 2 O formation in the soil48. The forest floor fluxes subsequently decreased in the dormant season, which was in accordance with our earlier findings48. The effect of soil VWC on N 2 O fluxes was most pronounced during the dormant season, when N 2 O consumption was observed in the soils with low VWC (0.21 m3 m−3; i.e. dry plot; Fig. 5d). This N 2 O consumption reduced the annual forest floor N 2 O emissions by 40% at the dry plot (Fig. 5d). Nevertheless, forest floor N 2 O consumption was occasionally observed at all the plots throughout the year (Fig. 1d). Even though the CO 2 emissions from the forest floor also were significantly lower in the dormant season compared to the vegetation season (Supplementary Fig. 1d, 4d), these were still detectable.

In summary, during the dormant season, the tree stems and forest floor remain sources of N 2 O at plots characterised by high soil VWC, whereas tree stems act as N 2 O sinks under moderately wet soil water conditions. During the vegetation season, however, the soil VWC does not affect the N 2 O emissions from either trees or forest floor. Hence, our results highlight the need for winter flux measurements in order to correctly estimate the overall N 2 O budget of boreal forests.

Boreal trees are net annual N 2 O emitters

All the tree species studied were net sources of N 2 O at the annual scale (Fig. 6). To the best of our knowledge, this is the first study reporting annual course of N 2 O fluxes in boreal trees to include winter measurements. Neither the stem nor the forest floor N 2 O emissions were significantly influenced by the soil VWC at the annual scale (Fig. 1). Therefore, measurements at the wet and moderately wet plots, where all the species were present, were merged to evaluate the tree species-specific fluxes at the annual scale. The measurements at the dry plot were not included into this comparison because only pine trees were present there. Spruce was the strongest emitter of N 2 O, with total emission per year of 0.91 mg N 2 O m−2 stem area and 2.4 g N 2 O ha−1 ground area, followed by pine (0.47 mg m−2 and 1.7 g ha−1) and birch (0.38 mg m−2 and 0.71 g ha−1) (Fig. 6). The forest floor emitted in total 9.4 mg N 2 O m−2 soil area per year (i.e. 93.9 g ha−1 per year), which is consistent with the annual N 2 O emissions of 8.8 mg N 2 O m−2 yr−1 estimated for the same forest during the years 2002–200348.

Fig. 6 Annual N 2 O fluxes in tree stems and forest floor. The fluxes are expressed per stem or soil surface area unit (a) and scaled up to unit ground area of boreal forest (b). The fluxes are expressed as medians (solid line) and means (broken line) of measurements at both wet and moderately wet plots together, as the N 2 O fluxes did not vary significantly between those plots at the annual scale. The dry plot was not included into this comparison of annual fluxes because only pine trees were available at this plot. The stem fluxes were measured from six trees per species (n = 6), the forest floor fluxes were determined at six positions (n = 6). The box boundaries mark the 25th and 75th percentiles. Statistically significant differences in annual fluxes among birch, spruce and pine at p < 0.05 are indicated by different letters above the bars. The contributions of stem fluxes to forest floor N 2 O fluxes (equal to 100%) are expressed as percentages of the forest floor flux Full size image

Based on the topographic wetness index (TWI) at the site, the dry plot represents 48%, the moderately wet plot 37%, and the wet plot 11% of the forest (remaining 4% accounting for standing water, Supplementary Fig. 5). Thus, we estimate that the annual emissions from the wet and moderately wet plots together represent ca 50% and the emissions from the dry plot ca 50% of the total forest fluxes, respectively. As we have demonstrated that the tree stem N 2 O fluxes are not controlled by soil water content at the annual scale, we confidently can conclude that the site type does not play a critical role in stem N 2 O fluxes.

Moreover, the differences in ecosystem level fluxes may result from tree species composition of the forest. As we found that spruce tree stems emitted significantly more N 2 O than did pine and birch stems, spruce-dominated forests are predicted to emit more N 2 O than pine- or birch-dominated forests. The contribution of tree species to the forest floor N 2 O emissions was relatively low, however, amounting to 2.5, 1.8, and 0.75% for spruce, pine, and birch, respectively (Fig. 6) when the certain representation of tree species at each plot (Table 1) was included in upscaling. In Finland, Scots pine is the dominant tree species, accounting for 78% of forest land area coverage, while only 15% is covered by Norway spruce49. Our finding of the N 2 O emissions from spruce trees can be important for the estimation of N 2 O budget not only in boreal forests but also in temperate forests of Central Europe, where spruce is widely grown in monoculture50. Spruce trees’ stronger capability to exchange N 2 O with the atmosphere may be related to their physiological activity. In our study, spruces had the highest projected leaf area per tree (88 m2 on average) among the tree species studied (birch 54 m2, pine 28 m2). Greater leaf area results in larger amounts of CO 2 assimilated and H 2 O transpired per spruce tree than per pine or birch tree. The greater physiological activity of spruce is further reflected in the higher annual sum of stem CO 2 efflux amounting to 0.867 kg CO 2 m−2 and 2303 kg ha−1, compared to 0.590 kg CO 2 m−2 (2140 kg ha−1) and 0.427 kg CO 2 m−2 (738 kg ha−1) for pine and birch trees, respectively (Supplementary Fig. 6). Ge et al.51 presented the same conclusion from their study of different boreal tree species. The variation in N 2 O emission rates among plant species also can result from plants’ effects on soil N 2 O production and consumption, which can themselves differ significantly among species, rather than from different transpiration rates or direct plant production of N 2 O in plant tissues52. Although deciduous tree species tend to increase soil N 2 O production more so than do conifers29,30, the effects of individual tree species are not uniformly presented among studies. Further research is therefore needed to understand the observed differences in N 2 O emission rates among tree species.

Table 1 Stand characteristics and tree biometric parameters Full size table

Lack of canopy level N 2 O flux measurements brings additional uncertainty in the forest ecosystem N 2 O budget. Based on our previous research, we have shown that the leaf emissions by pine trees could exceed those of stems by as much as 16 times13. We therefore expect that boreal tree species might contribute even more significantly to the forest N 2 O exchange. Although measurements of above-canopy N 2 O exchange in forest ecosystems using such micrometeorological techniques as eddy covariance, eddy accumulation, or flux gradient methods have been used only rarely53,54,55, these could improve our view in the future.

We have demonstrated that N 2 O emissions from tree stems are driven by physiological activity of the trees and by ecosystem activity, showing higher emissions during the active growing period and variation between uptake and emissions during the dormant season. Although our study may well be applicable to large upland forest areas in the boreal zone, which are typically N limited56, our findings may not apply directly in N-affected central European or American forests known to exhibit elevated soil N 2 O emissions due to higher soil N content and faster N turnover rates57,58,59. The N status of a forest directly influences soil N 2 O concentration, which has been shown to be a good proxy for N 2 O transport via the transpiration stream of trees8. Until more studies and process understanding emerge, the global strength of N 2 O emissions from trees will remain largely unknown and could possibly be estimated by, for example, adding a fixed percentage (e.g. 10%) to the forest floor N 2 O emissions to represent N 2 O emission from trees.