Energetic supply regulates heterotrophic nitrogen fixation along a glacial chronosequence
Introduction
Glaciers on the Tibetan Plateau and in its surroundings are experiencing shrinkage, with associated impacts on ecosystem functions and human livelihood (Huss et al., 2017; Yao et al., 2012). The newly exposed lands after glacier retreat are typically deficient in nitrogen (N), and the establishment of vegetation largely depends on N input through biological N fixation (BNF) (Chapin et al., 2016; Reed et al., 2011). BNF includes symbiotic N fixation and free-living N fixation (FLNF). Symbiotic N fixation occurs in the root nodules of N-fixing plants and is carried out by Rhizobia and Frankia bacteria that fix N in conditions well moderated by the plant host. FLNF is carried out by autotrophic or heterotrophic diazotrophs without the transfer of C from a formal plant host (Dynarski and Houlton, 2018). Free-living diazotrophs are a highly diverse group from a wide range of bacterial phyla (e.g., Proteobacteria, Firmicutes, Cyanobacteria) and methanogenic archaea, and they may survive in variable and dynamic environments (Smercina et al., 2019). In temperate areas, N-fixing plants are generally constrained to the early stage of ecosystem development (Menge et al., 2019), although some species may persist for centuries in secondary succession (e.g., green alder in upland forests throughout interior Alaska; Mitchell and Ruess, 2009). In contrast, FLNF is ubiquitous in different successional stages and climate zones and can be found in plant litter, soil, and in association with moss or lichen (Dynarski and Houlton, 2018; Reed et al., 2011). In different biomes, the average amount of N input from FLNF ranges from 1.0 to 15.0 kg N ha−1 year−1, which is comparable to or even greater than that from symbiotic N fixation (Cleveland et al., 2010; Reed et al., 2011). Specifically, FLNF represents an essential source of N input when symbiotic N-fixing plants are absent (Pérez et al., 2014). However, the changing patterns of FLNF during ecosystem succession after glacier retreat have received little attention, which limits our ability to adapt to a future world with more ice-free areas.
FLNF is catalyzed by nitrogenases that are metalloenzymes encoded by the N fixation (nif) genes. The nif genes include structural genes of the highly conserved nitrogenase complex and the genes required for nitrogenase biosynthesis and regulation (Dixon and Kahn, 2004). The synthesis and functioning of nitrogenases are affected by climate (e.g., light, temperature, moisture), soil properties (e.g., pH) and diazotroph community, and require an adequate supply of phosphorus (P), iron and molybdenum (or vanadium in the case of alternative nitrogenases) (Dynarski and Houlton, 2018; Reed et al., 2011; Smercina et al., 2019; Vitousek et al., 2002). In addition, nitrogenase-catalyzed N2 reduction is energetically expensive, and even more energy is needed to protect nitrogenase from inactivation by oxygen (O) (Smercina et al., 2019). Therefore, FLNF is tightly regulated by environmental factors mainly at the transcription level (Dixon and Kahn, 2004; Ryu et al., 2020). The well-studied N regulation networks of FLNF consist of two components including the N sensor proteins typically belonging to the PII superfamily and the nif-gene regulation system, which sense the N status and regulate the transcription of nif genes according to the N status (Dixon and Kahn, 2004; Leigh and Dodsworth, 2007). Nutrient addition experiments reveal that a sufficient N supply depresses FLNF because the uptake of fixed N is less costly than BNF (Dynarski and Houlton, 2018). However, a recent study has demonstrated increased FLNF rates in N-rich late-successional ecosystems compared with relatively N-poor early-successional ecosystems (Zheng et al., 2020). The authors highlight the role of the C:N ratio in explaining the variations in FLNF rates (Zheng et al., 2020). This stoichiometric view is in line with the findings that the N status-sensing PII proteins and the nif-gene regulation systems of some diazotrophs are coregulated by C and N signals (e.g., 2-oxoglutarate and glutamine) and that N signals override C signals to depress N fixation only in situations of N excess (Dixon and Kahn, 2004). In addition to the C:N ratio, C availability alone may be a major predictor of FLNF rates. This is because in contrast to symbiotic organisms, heterotrophic diazotrophs must find their own source of energy, typically by oxidizing organic molecules released during organic matter decomposition or released by other organisms (e.g., low molecular weight C compounds in plant root exudates) to perform the energetically expensive N fixation. Some previous studies report that the heterotrophic FLNF in litter and soil is limited by C quantity and quality (Jones and Bangs, 1985; Vitousek and Hobbie, 2000). However, whether C availability or C:N ratio is the major influencing factor of FLNF rates remains untested.
Another issue associated with the regulators of FLNF is to what degree the abundance of nif genes (gene potential) may determine FLNF rates. A recent study has demonstrated that the activities of C-degrading hydrolytic enzymes in soils are closely and positively related to their gene abundances (Trivedi et al., 2016), indicating the existence of constitutive expression for these hydrolase (Allison et al., 2005). However, nitrogenase activity may deviate from its gene potential because the expression of nif genes is tightly regulated by resource availability and not likely to be constitutive. Among different studies, FLNF rates and nifH gene (encoding Mo-nitrogenase) abundance have been reported to be positively related or unrelated (Brankatschk et al., 2011; Chen et al., 2019; Reed et al., 2011). The discrepancy may arise from the covariation between nifH gene abundance and resource availability. Therefore, it is necessary to conduct a multiple-variable analysis that clarifies how gene potential and resource availability influence FLNF rates.
In this study, we selected four sites (exposed since 2012, 1980, 1970 and 1930, respectively) along the ~130-year Hailuogou glacier chronosequence and a reference site with well-developed Podosols on the eastern Tibetan Plateau to investigate the FLNF rates in successional ecosystems after glacier retreat. Along the chronosequence and at the reference site, light conditions markedly decrease as canopy closes and resource availabilities also vary with soil depth and along successional stages (Wang et al., 2020c). The potential rates of FLNF (nitrogenase activity) in litter and soil were measured in the field (with ambient light) and in the lab (without light). We supposed that the rates measured in the lab were from heterotrophic FLNF because autotrophic FLNF relies on photosynthesis for energy acquisition (Pérez et al., 2017; Stal, 2012). We hypothesized that (1) a shift from photosynthetic FLNF to heterotrophic FLNF would occur as the canopy closes, due to the decreased light intensity and increased resource availabilities associated with plant litter and root exudates; (2) C availability, rather than N or C:N ratio, is the major factor controlling the heterotrophic FLNF rates in the relatively N-poor ecosystems after glacier retreat. In addition, the effect of gene potential on heterotrophic FLNF rates may be weak because the expression of nif genes is tightly regulated by resource availability and is not likely to be constitutive.
Section snippets
Study area
The ~130-year Hailuogou glacier chronosequence is located in the Gongga Mountains (Mt. Gongga), eastern Tibetan Plateau (Fig. S1). The chronosequence extends from the end of the glacier (~3000 m above sea level (a.s.l.)) to the northeast for less than 2 km, with an elevation decrease of less than 100 m. We selected four sites along the chronosequence and a reference site in a mature forest that is less than 2 km away from the chronosequence to represent five successional stages (Fig. S1). The
Nitrogenase activity
At Stage 1, the S layer showed significantly higher potential FLNF rates (represented by nitrogenase activity) when measured in the field than in the lab (Fig. S2; samples collected in June and August). At Stages 2–5, the Oi-layer FLNF rates measured in the field were similar to or lower than those measured in the lab (Fig. S2; samples collected in June and August). The subsurface-soil (SS layer at Stage 1; Oe, Oi and A layers at Stages 2–5) FLNF rates measured in the field were similar to
Patterns of potential FLNF rates during primary succession
Light represents a key regulator of photosynthetic FLNF but likely does not affect heterotrophic FLNF (Pérez et al., 2017), thus we checked if a shift from photosynthetic FLNF to heterotrophic FLNF may occur as canopy closes during plant succession. In the S layer at Stage 1, markedly higher FLNF rates were measured in the field (with ambient light) than in the lab (without light) (Figs. 1 and S2). This indicates the dominance of photosynthetic FLNF in the S layer, as supported by the
Conclusions
Several years after glacier retreat, photosynthetic cyanobacteria in biological soil crusts contributed significantly to FLNF, while heterotrophic N-fixing microorganisms in the litter layer played an important role in FLNF at later successional stages. We found a close relationship between C availability and heterotrophic FLNF rates, suggesting strong energetic controls of heterotrophic FLNF in newly exposed ecosystems after glacier retreat. Thus, in addition to NPP, MAP and C:N ratio as
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
We thank Editor Kate Orwin and two anonymous reviewers whose insightful comments greatly improved the manuscript. This research was supported by the National Natural Science Foundation of China (41701288 and 41630751) and the Youth Innovation Promotion Association, Chinese Academy of Sciences (2017424) and CAS “Light of West China” Program, Chinese Academy of Sciences.
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