Molybdenum threshold for ecosystem scale alternative vanadium nitrogenase activity in boreal forests

Widespread Presence and Activity of Alternative V-Nase in Peltigera Cyanolichens.

We detected the presence and activity of alternative V-Nases in a majority of the 104 colonies of Peltigera cyanolichen (Fig. 1 and Table 1) collected from 5 sites across a 600-km latitudinal transect within the boreal biome. Genes for V-Nase (vnfDG and vnfN) in the Nostoc cyanobacterial partner were identified in 28 of 30 cyanolichens representative of the diversity of our sampling (Table 1). Every Peltigera species typical of boreal forests (e.g., P. aphthosa, P. malacea, P. neopolydactyla s.l., P. scabrosa; Table 1) showed the presence of vnf genes. The 2 lichens samples for which we did not detect vnf genes are from Peltigera species found mainly in temperate regions (i.e., P. leucophlebia, P. canina; Table 1). Mo-Nase genes were detected in all tested samples. These results, consistent with earlier reports (22, 26, 27, 30), indicate that alternative V-Nase genes are a common feature of Nostoc found in Peltigera thalli at high latitudes.

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Fig. 1.

Presence and activity of V-Nase in boreal cyanolichens along a 600-km transect in eastern Canada. The black stars show the approximate location of the 5 sampling sites (n = sample size per site). Ringed pie-charts summarize the number of samples showing positive ethane (blue), ISARA (green), or both ISARA and ethane signals (yellow), the number of samples showing no V-Nase activity (dark gray), and the number of samples below the detection limit of either method (N.D., light gray). Numbers within the pie charts indicate the number of individual thalli in which vanadium nitrogenase genes were detected over the total number of samples tested. Molybdenum nitrogenase genes were detected for all 30 samples tested.

To elucidate the contribution of alternative Nases to total cyanolichen BNF, we used 2 independent methods (27, 28) that make use of the acetylene reduction assay (31) to detect isoform-specific signatures. In the isotopic acetylene reduction assay (ISARA, ref. 27), the characteristically low ¹³C fractionations of acetylene reduction to ethylene (¹³ε_AR = δ¹³C_acetylene − δ¹³C_ethylene) by alternative Nase isoforms (e.g., V-Nase) compared to those by Mo-Nases (¹³ε_Mo = 13.2–14.7‰_, ¹³ε_V = 7.5–8.8‰; ref. 27) can be used with measurements of sample ¹³ε_AR to calculate the contribution of each isoform to total BNF activity. In the ethane formation method (28), alternative Nase activity is detected based on the production of ethane as a side product during reduction of acetylene by the enzyme. The ratio of ethane to ethylene (negligible for Mo-Nase) in a given sample thus enables isoform-specific BNF activity to be estimated (ref. 28 and SI Appendix, Fig. S1). Both methods require samples, such as cyanolichens, with high levels of BNF activity. For the ∼75% fraction of cyanolichen samples with sufficient BNF activity (Fig. 1 and Table 1), alternative Nase activity was detected in 56% (31 of 55) of these thalli based on a low ISARA ¹³ε_AR value (<13.2‰; ref. 32), and in 49% (34 of 69) based on a high ethane ethylene ratio (i.e., >0.1%; SI Appendix, Fig. S1). More samples showed alternative Nase activity in the 3 northern sites (sites III–V, Fig. 1), with over 50% of the samples demonstrating a positive signal with at least one of the methods. Since Fe-only Nase genes have not been found in any cyanobacteria genomes to date (17), we attribute all alternative Nase activity in our study to the V-Nase isoform.

Ecosystem-Scale Contribution of Alternative V-Nase Activity in Peltigera Cyanolichens.

We investigated the spatial distribution of V-Nase activity in Peltigera lichens by analyzing samples collected during the early growing season (May, June) from multiple sites (sites I–V) spanning a 600-km latitudinal transect (Fig. 1). To elucidate temporal changes in V-Nase BNF, we also collected and analyzed samples at site IV throughout the growing season (late May to late September). Both the ISARA and ethane methods showed an increase in alternative Nase activity northwards along the transect and during the growing season at site IV (Fig. 2 A–F). The spatial pattern is shown by decreases in the median value of ¹³ε_AR (Fig. 2A) from a Mo-Nase only signal at the southernmost site (site I, ¹³ε_AR = 13.8‰) to values indicative of alternative Nase activity at higher latitude sites (sites II–V, ¹³ε_AR < 13.2‰, the lower bound of ¹³ε _AR for Mo-Nases; ref. 27). Based on ISARA, we estimate that 25–30% of N₂ reduction activity is due to V-Nase across sites II, III, IV, and V in May (SI Appendix, Table S1). Increases in V-Nase contributions to BNF at site IV (from ∼0 to 71% of N₂ reduction) during the growing season are shown by a steady decrease in median ¹³ε_AR values (from ∼14.5‰ to 11.5‰, Fig. 2B). Higher ratios of ethane to ethylene northwards along the transect provide further evidence of alternative Nase activity. Qualitatively consistent with ISARA estimates, we find that ∼10–20% of BNF can be attributed to V-Nase in samples from northern sites III, IV, and V in May (Fig. 2C and SI Appendix, Table S1) as well as a clear contribution of V-Nase to BNF at site IV from June to September (26–47%, Fig. 2D and SI Appendix, Table S1). Because ISARA and ethane methods are subject to different biases (SI Appendix, Methods), we use the average of the 2 proxies as the best estimate of V-Nase BNF. We find that alternative V-Nase accounted for 20–35% of total BNF by Peltigera cyanolichens collected in the northern part of the transect (beyond 48°N) in the early growing season (May, June). Given the increase in V-Nase activity during the summer at site IV (Fig. 2 E and F and SI Appendix, Table S1), our results suggest that V-Nase contributions at northern sites have increased to 50% or more by the end of the growing season.

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Fig. 2.

Alternative V-Nase activity and Mo thalli content along the south–north transect described in Fig. 1 (A, C, E, and G) and growing season at site IV (B, D, F, and H). The boxplots show the ISARA carbon isotopic fractionations (A and B), ethane productions (C and D), and contributions of alternative V-Nase to the total BNF activity calculated as the average of both methods when available (E and F). Molybdenum contents of the thalli are shown for comparison (G and H). In A–D, the right axis represents the estimated contribution of V-Nase to BNF (note the direction of axis). Filled symbols represent values outside the whiskers, i.e., 1.5 interquartile range over the upper quartile or below the lower quartile. When filled symbols are absent, the whiskers represent the full range of the data. The horizontal colored boxes in A–D show the range of Mo-only signal for each proxy (ethane, <0.1% of C₂H₄; ISARA, ¹³ε_AR > 13.2‰).

The measurement of significant and widespread contributions by alternative V-Nase to cyanolichen BNF confirms prior genomic (17, 22), isotopic (26, 27), R ratio (33), and micronutrient evidence (25, 26) indicating that alternative Nases are more important for environmental BNF than previously thought. Estimates of V-Nase contribution to BNF across all sites based on ISARA and ethane methods (individually or averaged) ranged from 0 to 74% (Fig. 2 and SI Appendix, Table S1). These results are consistent with previous estimates from individual cyanolichen thalli collected in southern Québec and northern Sweden (0–60%, refs. 26 and 27). Our findings that V-Nase contributions to BNF increase over the growing season are supported by recent literature suggesting Mo limitation is seasonal in cold temperate and boreal forests (34, 35).

Large contributions of V-Nase to BNF have important consequences for BNF rates assessed using acetylene reduction methods and, thus, for N budgets. The ratio of the acetylene reduction rate to N₂ fixation (N₂ reduction) rate is higher for Mo-Nase (R ratio = 3–4) than V-Nase (R ratio < 2; ref. 33). Our data indicate that the sole use of the acetylene reduction assay method to assess N₂ fixation rates, i.e., without considering alternative Nase activity, results in large underestimation of BNF rates (by up to ∼30% during the late period of the growing season at site IV). This finding is particularly relevant to cold, N-limited high latitude ecosystems, such as boreal forest and arctic tundra, where BNF by cryptogamic covers sustains an important part of N input (11, 12).

Mo Threshold for Alternative V-Nase Contribution in the Boreal Forest.

Because Mo levels have been identified as the primary factor controlling V-Nase activity in laboratory studies (18⇓–20), we assessed the role of Mo availability as a driver of cyanolichen V-Nase activity. We found that the Mo content of Peltigera thalli varied across latitude and during the growing season (Fig. 2). Mo deposition on the forest floor, typically assessed with measurements of cryptogam elemental composition (36), was highest at site I (median content of Mo in Peltigera thalli = 300 ng_Mo·g_thallus⁻¹) then decreased northwards to 65 and 90 ng_Mo·g_thallus⁻¹ at site IV and V, respectively (Fig. 2G). These data indicate higher metal exposure in the southernmost portion of the boreal forest, in agreement with a previous large-scale study of metal deposition in northern Québec (29). Mo content also varied seasonally: At site IV, Peltigera thalli Mo levels (Fig. 2H) were found to be the highest in May, with a median value of 230 ng_Mo·g_thallus⁻¹, followed by a steady decrease down to a baseline of 50–100 ng_Mo·g_thallus⁻¹ from July to September.

As expected, average Mo contents of lichen thalli were inversely related to average V-Nase activities along the sampling transect and the growing season, suggesting that Mo drives spatial and temporal variation of V-Nase activity (Fig. 2 E–H). Sites with high median and large ranges in lichen Mo content (Fig. 2 G and H) exhibited the lowest level of V-Nase activity as measured by both ISARA and ethane methods (i.e., site I samples in May 2017, site IV samples in May 2016; Fig. 2 A–F). Conversely, site IV and V samples in 2017 exhibited low Mo and high V-Nase activity. Interestingly, substantial V-Nase activity (>15% of N₂ reduction activity) was only found at sites where the median Mo content was lower than ∼200 ng_Mo·g_thallus⁻¹ (Fig. 2), suggesting the existence of a Mo threshold for V-Nase activity.

Because both Mo thallus content and V-Nase contribution exhibited large intrasite variability (Fig. 2), we determined whether the relationship between Mo content and V-Nase BNF could be better explained at the level of individual thalli. We found a clear negative, nonlinear relationship (Fig. 3, log–log regression) between Mo content and V-Nase contribution to BNF as estimated using the ISARA (Fig. 3A; R² = 0.26, P < 0.001, n = 58) and ethane methods (Fig. 3B; R² = 0.41, P < 0.001, n = 72). The vast majority of lichen samples showing V-Nase activity contained less than ∼250 ng_Mo·g_thallus⁻¹ (see the vertical lines in Fig. 3), indicating the existence of a Mo threshold for V-Nase activity. Indeed, the highest V-Nase contributions to BNF (up to 80%) were identified in lichens with the lowest Mo content (30 ng_Mo·g_thallus⁻¹). With a few exceptions, no substantial V-Nase contributions (i.e., within the margin of error as shown by horizontal colored boxes in Fig. 3) were found in lichens with high Mo content (400 ng_Mo·g_thallus⁻¹).

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Fig. 3.

Relationship between Mo content in Peltigera and contribution of V-Nase to BNF in individual thalli. Each point represents an individual thallus from either the 2017 (circle) and 2016 (square) field seasons. Vanadium nitrogenase contributions were separately estimated based on the ethane (A, n = 58 samples) and ISARA methods (B, n = 72 samples). Linear regression was performed on the log–log transformed data and results were back-transformed in the original space. Dashed lines show 95% confidence intervals and gray shadowed areas show 95% prediction intervals for this regression. Horizontal colored boxes derive from ethane and ISARA values for which V-Nase BNF contributions are uncertain (ethane, <8% V-Nase; ISARA, <34% V-Nase). Vertical red lines show the estimated Mo concentration threshold for the onset of V-Nase activity based on visual analysis (Mo_threshold = 250 ng_Mo·g_thallus⁻¹).

These results provide compelling ecosystem-scale evidence for the use of alternative V-Nase as a supporting enzyme that sustains BNF under Mo-limited conditions.

Contribution of Alternative V-Nase to Terrestrial N Input.

Interestingly, the 250 ng_Mo·g_thallus⁻¹ threshold value reported here for the onset of alternative V-Nase activity in Peltigera thalli falls within the range of previous threshold estimates (200–300 ng_Mo·g_{leaf litter}⁻¹) for Mo limitation of BNF in tropical leaf litter, as assessed with Mo amendment experiments (37). This striking agreement suggests a “global” threshold for Mo limitation of free-living BNF across organisms and biomes as well as similarities in the biogeochemical controls on Mo availability to these organisms across ecosystems.

The results imply that the Mo content of cyanolichens can be used to predict the contribution of V-Nase to BNF. Indeed, lichen Mo contents (≤ 250 ng_Mo·g_thallus⁻¹) correctly predicted V-Nase activity in 95% of the samples that showed V-Nase activity based on ISARA or ethane methods (n = 82; SI Appendix, Table S2). Our literature survey of Mo contents in Peltigera lichen thalli across the circumboreal belt (25, 26) confirms that most of the boreal area is exposed to relatively low levels of Mo through atmospheric deposition (Fig. 4). The average Mo concentration at sites within the Canadian–European boreal region (Alberta, northern Québec, and northern Sweden) falls below the threshold of 250 ng_Mo·g_thallus⁻¹. Even in sites that show higher Mo content due to their proximity to anthropogenic activity (median value from 250 to 500 ng_Mo.g_thallus⁻¹ in Russia, Alaska, southeastern Canada; Fig. 4), several individual thalli exhibited Mo levels below the threshold, indicating that V-Nase might still contribute to BNF in these areas (see histograms in Fig. 4). Indeed, we found the presence of V-Nase genes and evidence for V-Nase activity in Peltigera samples collected across the circumboreal belt (32, 37⇓–39), including in human-impacted areas (32). These results imply that the reliance of boreal ecosystems on V-Nase N input would have been higher in preindustrial times when atmospheric deposition of metals and fixed N, which increase with anthropogenic activities (40, 41), were lower. Recent increases in anthropogenic pollutants enriched in Mo and/or V, such as tar sands (41, 42), could provide new fluxes of Mo and V to natural environments. Conversely, successful pollution control policies (e.g., the Clean Air Act in the United States), which have led to recent declines in atmospheric deposition of heavy metal pollutants and reactive N (43), may help to maintain Mo limitation of BNF in high latitude N-limited forests and increase the need for V-Nase BNF. Interestingly, previous studies on cyanolichens (25, 26) suggest that V might also be limiting BNF in areas with low levels of metal deposition, such as in northeastern Canada (29).

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Fig. 4.

Putative biome scale activity of vanadium nitrogenase in the circumboreal belt. Map of the circumboreal biome with the geographic position and Mo content of lichen thalli from available literature (25, 26, 29) and from this study. Light gray, close-up map (Left) of the circumboreal area outlined by dashed box shows the location and median Mo content of samples from northeastern North America (Québec). The histograms indicate the distribution of Mo content in lichen thalli at each location on the globe, with colors indicating median Mo content of thalli. The red line indicates the Mo content threshold for the onset of V-Nase activity.

Our findings that V-Nase supplements cyanolichen BNF under Mo limitation in boreal forests may be an example of a general strategy of free-living and cryptogam associated N₂ fixers, which cannot easily benefit from the nutrient mining potential of tree roots like symbiotic nodule associated N₂ fixers (16, 34, 35). Nostoc cyanobacteria, the diazotrophic constituents of Peltigera cyanolichens, represent a globally distributed genus in terrestrial environments (e.g., biocrust, bryophytes, soils, freshwater; refs. 38 and 43⇓–45). Genes for V-Nase have been found in the genome of Nostoc associated with cyanolichens and bryophytes from temperate to tropical areas (22, 46). In addition, taxonomically diverse V-Nase genes have also been identified in other substrata, including wood mulch, sediment from mangroves and salt marshes, and forest soils (17, 21, 33), suggesting that a variety of microorganisms (e.g., Proteobacteria, Firmicutes) rely on alternative BNF for N acquisition. Because Mo limitation of BNF is a common feature of terrestrial ecosystems (16), where cryptogamic covers are estimated to contribute half of terrestrial nitrogen fixation (9), our research calls for a broad reevaluation of the importance of alternative Nases to N cycles and budgets.