Niche differentiation of bacteria and fungi in carbon and nitrogen cycling of different habitats in a temperate coniferous forest: A metaproteomic approach

https://doi.org/10.1016/j.soilbio.2021.108170Get rights and content

Highlights

  • Habitats in a temperate coniferous forest differ in their protein composition.

  • Bacteria dominated N cycling while fungi contributed more to C cycling.

  • CAZymes in roots and litter targeted mostly plant structural biopolymers.

  • Ammonium assimilation in litter/roots and nitrification in rhizosphere/soil.

Abstract

Temperate coniferous forests sustain the highest levels of biomass of all terrestrial ecosystems and belong to the major carbon sinks on Earth. However, the community composition and its functional diversity depending on the habitat have yet to be unveiled. Here, we analyzed the proteomes from litter, plant roots, rhizosphere, and bulk soil in a temperate coniferous forest at two time points to improve the understanding of the interplay between bacterial and eukaryotic communities in different habitats. Our metaproteomic approach yielded a total of 139,127 proteins that allowed to differentiate the contribution of microbial taxa to protein expression as well as the general functionality based on KEGG Orthology in each habitat. The pool of expressed carbohydrate-active enzymes (CAZymes) was dominated by fungal proteins. While CAZymes in roots and litter targeted mostly the structural biopolymers of plant origin such as lignin and cellulose, the majority of CAZymes in bulk and rhizosphere soil targeted oligosaccharides, starch, and glycogen. Proteins involved in nitrogen cycling were mainly of bacterial origin. Most nitrogen cycling proteins in litter and roots participated in ammonium assimilation while those performing nitrification were the most abundant in bulk and rhizosphere soil. Together, our results indicated niche differentiation of the microbial involvement in carbon and nitrogen cycling in a temperate coniferous forest topsoil.

Introduction

A large share of the Earth's carbon (C) resides in living trees, forest vegetation and soil microbial biomass of temperate coniferous forests (Pan et al., 2011). Understanding their functional responses to global change is important for predictive climate modelling as they act as both C sources and sinks. The C cycling in forest topsoil is highly integrated within the biosphere. Its complex dynamics are influenced by both soil community functions and a heterogeneous trophic environment. Soil microbes contribute to a variety of ecosystem functions such as the decomposition of organic matter, tree root symbiosis and pathogenicity. Microbes inhabiting litter, on the other hand, depend on complex litter-bound nutrients recycled by microbial decomposition that is partly mineralized and partly transformed into organic matter, which accumulates in the forest floor (Baldrian, 2017). Plant root symbionts, especially mycorrhizal fungi, act as important regulators of plant productivity and are responsible for the acquisition of limiting nutrients such as nitrogen (N) or phosphorus (Van Der Heijden et al., 2008), especially in nutrient-poor ecosystems (Franklin et al., 2014). Mycorrhizal fungi dominate the fungal community in the coniferous forest soils below the litter layer (Lindahl et al., 2007) and represent potentially the largest pool of microbial biomass (Ekblad et al., 2013). By extending the tree roots by their hyphae, they mediate the flow of tree-derived C through its roots into the soil, which can account for more than 30% of total primary production in these ecosystems (Clemmensen et al., 2013; Franklin et al., 2014). The topsoil clearly represents the major compartment of C storage and microbial activity in coniferous forests but understanding the processes of C and nutrient cycling is, however, complicated due to its spatial heterogeneity resulting from the existence of four habitats: litter, plant roots, rhizosphere, and bulk soil that are distinct in many respects (Baldrian, 2017). In addition to the fact that soil and rhizosphere contains smaller amount of organic matter than litter and roots, roots represent a habitat where the bulk of the plant biomass is living and thus not readily degraded. Furthermore, spruce litter and roots also differ in the content of major biopolymers: brown spruce needles contain around 32% lignin, 29% cellulose and 21% hemicelluloses (Johansson, 1995) while fine spruce roots (<2 mm) are reported to contain more lignin (34%) and less cellulose (20%) and hemicelluloses (9% (Hobbie et al., 2010);). Therefore, the application of the same analytical tools to all these habitats is the best option to understand and to relate their functions in ecosystem processes that reflect their properties.

Specific functions of the topsoil organisms can be statistically inferred based upon the homology of genes, transcripts or proteins that are present to experimentally characterized genes and proteins in known organisms. This so-called ortholog annotation is performed by the comparison to phylogenomic databases, covering either a wide range of functional classes, e.g., KEGG Orthology (Kanehisa et al., 2016a, Kanehisa et al., 2016b) or more specific subsets, such as the carbohydrate-active enzymes (CAZymes) (Lombard et al., 2014) involved in C cycling. Processes of C cycling are closely related to N cycling as their ratio can be used to identify the origin of a source (Müller, 1977; Ishiwatari and Uzaki, 1987; Prahl et al., 1994). The theory of ecological stoichiometry suggests that strictly homeostatic organisms have low nitrogen use efficiency (NUE) but high carbon use efficiency (CUE) at low substrate C/N-ratios (Sterner and Elser, 2002). By contrast, these organisms are expected to lower their CUE while increasing their NUE at high substrate C/N-ratios (Mooshammer et al., 2014). Logically, there exists a threshold elemental ratio (TER) that defines the elemental ratio at which the metabolic control of an ecological system switches from C limitation to N limitation (Urabe and Watanabe, 1992; Anderson and Hessen, 1995). The result is an inverse relationship between C and N cycling separated by the TER (Mooshammer et al., 2014) that may yield a different set of partaking microbes (Starke, 2017). In coniferous forest soils, a variety of C sources of different complexity are decomposed with a vast arsenal of glycoside hydrolases (GHs) that hydrolyze glycosidic bonds (Lombard et al., 2014). The functional classification comprises families of structurally-related catalytic and carbohydrate-binding modules or functional domains of enzymes that degrade, modify or create glycosidic bonds (Lombard et al., 2014), and are thus important for the breakdown of the different components of biomass: cellulose, hemicellulose and pectin from plant, chitin from fungal and peptidoglycan from bacterial cell walls. Importantly, however, members of the same CAZymes family can catalyze different reactions and their family membership may not sufficiently indicate the targets of their activity (López-Mondéjar et al., 2016) as seen in the family GH5. In addition to GHs, CAZymes include glycosyl transferases (GTs), carbohydrate esterases (CEs) and polysaccharide lyases (PLs) involved in the decomposition processes as well as auxiliary activities (AA) that cover redox enzymes acting in conjunction with CAZymes. The AA class is key for the decomposition of lignin by oxidative enzymes (Levasseur et al., 2013). On the other hand, N is available in the environment in different chemical forms: organic nitrogen, ammonium, nitrite, nitrate, nitrous oxide, nitric oxide, and inorganic nitrogen gas. Several enzymes predominantly produced by microorganisms are involved in their interconversions during assimilation, denitrification, nitrification or nitrogen fixation (Simon and Klotz, 2013; Sparacino-Watkins et al., 2014). For the global N budget, nitrogen fixation in natural ecosystems (Vitousek et al., 2013) as well as agricultural systems (Fowler et al., 2013), the emission of oxidized N forms from soil (Pilegaard, 2013) and of ammonium in terrestrial ecosystems (Sutton et al., 2013) were previously deemed important.

The use of metaproteomics became more and more popular to study how microbes contribute to soil ecosystem services (Von Bergen et al., 2013) as proteins not only provide both functional and taxonomic information (Hettich et al., 2013; Von Bergen et al., 2013; Wilmes et al., 2015), but also represent the catalysts of important biochemical reactions (Wong, 2009). In favor of using proteins are: (i) the extracellular activity of soil proteins that can persist and remain active through the stabilization by humic substances and clay (Burns et al., 2013) whereas up to 40% of DNA (Carini et al., 2016) and 6% of RNA (Papp et al., 2018) were reported to derive from dead cells; (ii) the limitations of nucleic acids sequencing such as quantitative accuracy (Feinstein et al., 2009; Hungate et al., 2015), which may result in biased proportions of community members or functions; (iii) the difference between mRNA and protein levels depending on the temporal scale, on the complexity of the biological system and on the type of perturbation (Liu and Aebersold, 2016). Methodological advances, such as two-dimensional liquid chromatography (Callister et al., 2018) and site specific databases from sequencing approaches, now allow for a high protein identification rate that makes it possible to detect proteins with low relative abundance, such as CAZymes or proteins related to N cycling. Even though they are essential for the ecosystem functioning, they make up only a small part of the proteome that is dominated by structural proteins and proteins participating in the central metabolism.

In this study, we used a metaproteomic approach combining a site specific database comprising metagenomes, metatranscriptomes and fungal genomes together with two-dimensional liquid chromatography (Callister et al., 2018) to unveil the taxonomic and functional composition of the metaproteome in a temperate coniferous forest. In addition to describing the general functionality using KEGG Orthology, we have specifically addressed CAZymes that are involved in C cycling and proteins involved in N cycling. Four contrasting habitats – litter, plant roots, rhizosphere, and bulk soil, were compared to unveil habitat specific functions. We hypothesized that the utilization of plant biopolymers performed by fungi will be highest in litter, while components of microbial biomass targeted largely by bacteria (Lladó et al., 2017; López-Mondéjar et al., 2018) will be more important in soil and rhizosphere. Due to the high C/N-ratio and the high share of biomass-derived C in litter, we also expected that the incorporation of ammonia will be higher in litter compared to rhizosphere and soil where the share of inorganic N is higher and the C/N-ratio is lower (Baldrian et al., 2012).

Section snippets

Study area and sample collection

The study area was located at high altitudes (1170–1200 m) of the Bohemian Forest mountain range (Central Europe; 49°02 N, 13°37 E) and was covered by an unmanaged Norway spruce (Picea abies (L.) H. Karst.) forest. The mean annual temperature was 5 °C and the mean annual precipitation was 1000 mm. The understory was either missing or composed of grasses (Avenella, Calamagrostis), bilberries (Vaccinium) and mosses. The same study area was explored previously to identify the total and active

Chemistry of litter, roots, and soil

Litter showed a significantly higher pH (P = 0.0055 with H2O and 0.0171 with KCl) than soil (Table 1). Total nitrogen was significantly higher in litter as compared to soil (P = 0.0003) where both ammonium and nitrate were comparable between the two habitats. Both total carbon (P = 0.0002) and organic carbon (P = 0.0011) was significantly higher in litter while carbonate carbon was higher in soil (P = 0.0726). Calcium (P = 0.0074), magnesium (P = 0.0020), and potassium (P = 0.0054) were all

Remarks to the metaproteomic approach

Generally, metaproteomic approaches yield thousands of proteins per sample (Kleiner, 2019). However, with the relative CAZymes abundance of up to 5% (Žifčáková et al., 2017) only tens to hundreds of proteins identified as CAZymes will be found per sample. This is the reason why protein abundances for CAZymes have not been reported until now. The total of 139,127 proteins recovered from four forest habitats in our study make it possible to generate sufficient absolute numbers of CAZymes

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

This work was supported by the Czech Science Foundation (18-25706S and 20-02022Y), by the Ministry of Education, Youth and Sports of the Czech Republic (LTT17022). A portion of this research was performed under the Facilities Integrating Collaborations for User Science (FICUS; #49499) program and used resources at the DOE Joint Genome Institute and the Environmental Molecular Sciences Laboratory (grid.436923.9), which are DOE Office of Science User Facilities. Both facilities are sponsored by

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