Phosphate addition increases tropical forest soil respiration primarily by deconstraining microbial population growth

https://doi.org/10.1016/j.soilbio.2018.11.026Get rights and content

Highlights

  • P amendment increased tropical soil CO2 respiration, except for the most P-rich soils.

  • Genes involved in cell replication were relatively more transcribed with P amendment.

  • A multi-omics approach inferred that the trend of enhanced growth was community-wide.

  • Metabolisms for α-glucosyl polysaccharide storage were more abundant in low-P soils.

  • P amendment enhanced α-glucosyl substrate use; response often involved phosphorylases.

Abstract

Tropical ecosystems are an important sink for atmospheric CO2; however, plant growth is restricted by phosphorus (P) availability. Although soil microbiota facilitate organic P turnover and inorganic P mobilization, their role in carbon-phosphorus coupled processes remains poorly understood. To advance this topic, soils collected from four sites representing highly weathered tropical soils in the El Yunque National Forest, Puerto Rico were incubated with exogenous PO43− under controlled laboratory conditions. P amendment increased CO2 respiration by 14–23% relative to control incubations for soils sampled from all but the site with the greatest total and bioavailable soil P. Metatranscriptomics revealed an increase in the relative transcription of genes involved in cell growth and uptake of other nutrients in response to P amendment. A new methodology to normalize gene expression by population-level relative (DNA) abundance revealed that the pattern of increased transcription of cell growth and division genes with P amendment was community-wide. Soil communities responsive to P amendment possessed a greater relative abundance of α-glucosyl polysaccharide biosynthesis genes, suggestive of enhanced C storage under P-limiting conditions. Phosphorylase genes governing the degradation of α-glucosyl polysaccharides were also more abundant and increased in relative transcription with P amendment, indicating a shift from energy storage towards growth. Conversely, microbial communities in soils nonresponsive to P amendment were found to have metabolisms tuned for the phosphorolysis of labile plant-derived substrates, such as β-glucosyl polysaccharides. Collectively, our results provided quantitative estimates of increased soil respiration upon alleviation of P constraints and elucidated several underlying ecological and molecular mechanisms involved in this response.

Introduction

Despite covering <10% of the global land surface, tropical ecosystems account for one third of terrestrial net primary productivity and harbor one fourth of terrestrial biosphere carbon (C) (Bonan, 2008). Tropical ecosystems also play a major role in offsetting anthropogenically-emitted carbon dioxide (CO2) through enhanced plant uptake (Bala et al., 2007). However, CO2 uptake is limited by the availability of other essential nutrients, especially phosphorus (P) (Oren et al., 2001; Beedlow et al., 2004; Cleveland et al., 2011). In many highly weathered tropical soils, P is often regarded as the primary limiting nutrient for biological activities (Vitousek, 1984; Tanner et al., 1998). Belowground microbial activities are vital to the scavenging of inorganic P and for the turnover of P-containing plant and microbial detritus, thereby increasing P availability for both plants and microorganisms. While several previous studies have focused on how P availability constrains aboveground communities and C cycling, microbial activities involved in P cycling and soil organic carbon (SOC) turnover (CO2 release) remain much less understood (Turner and Wright, 2014).

P content has been found to regulate microbial activities in tropical forests (Cleveland et al., 2002; Cleveland and Townsend, 2006), and prior studies have shown that alleviating P constraints can result in enhanced soil microbial growth and activity. For instance, in a decade-long nutrient-fertilization experiment in Panama, soil microbial biomass carbon (MBC) responded to added P but not to other nutrients (Turner and Wright, 2014). While a 25% increase in fine litterfall from added P could have contributed to the observed increase in MBC (Wright et al., 2011), another study conducted at an adjacent site found no change in MBC when soil was supplemented with doubled plant litter inputs (Sayer et al., 2012). Therefore, the increased MBC in these studies is presumably attributable to direct microbial P usage. However, a mechanistic understanding of how the added P promoted microbial growth and which gene(s) can serve as biomarkers for this response remains elusive. Such biomarkers could be important for improved modeling and managing of P-limited tropical soil ecosystems.

Given that other nutrients are present in excess or are otherwise biologically obtainable, P-limitation could constrain microbial biomass directly by limiting the de-novo biosynthesis of P-containing ‘cell infrastructure components’, such as DNA, RNA, glycerophospholipids, ATP, and NADPH. Cultivated microorganisms adapted to P limitation are known to suppress their cell replication mechanisms while exhibiting a state of dormancy induced by P starvation (Drebot et al., 1990). During states where nutrients such as P are limiting and organic matter substrates are available in excess, microbiota have been shown to biosynthesize and accumulate α-glucosyl polymers such as glycogen and trehalose to serve as readily accessible energy sources for when nutrient conditions improve (Zevenhuizen, 1966; Lilie and Pringle, 1980). However, it remains unclear if the previous findings based mostly on lab-scale experiments with microbial isolates apply to in-situ soil conditions involving highly diverse substrates and complex traits of microbial communities, or what other traits low-P-adapted soil microorganisms possess.

The high diversity of soil communities compared to other environments (Rodriguez and Konstantinidis, 2014) and large degree of compositional heterogeneity makes consistent, reproducible patterns difficult to ascertain. To address these challenges, ‘multi-omics’ approaches have the potential to improve ecological interpretations and provide a more resolved quantification of microbial response compared to DNA-only approaches (Hultman et al., 2015; Coolen and Orsi, 2015; Mackelprang et al., 2016). For instance, by relating transcriptional activity to in-situ DNA abundance, immediate transcriptional responses can be evaluated at greater resolution and at both the whole-community and the individual genome/population levels. Metatranscriptomic or multi-omics approaches can also uncover shifts in microbial activities that aren't well reflected by corresponding gene frequency changes in metagenomes (DNA level), particularly in cases of sudden changes or short-lived pulses that are not accompanied by growth/replication (Singer et al., 2017).

In this study, we evaluated how highly weathered tropical soils respond acutely to the addition of a readily available P source. Triplicate soil samplings were collected from four locations in the El Yunque National Forest in Puerto Rico. Short-term laboratory incubation experiments were employed to relate heightened soil respiration from the amendment of exogenous phosphate to changes in microbial community structure and transcriptional activity assessed by metagenomics and metatranscriptomics, respectively, and to enzyme activities involving soil organic carbon (SOC) and soil organic phosphorus (SOP) mineralization. Our goal was to address the following hypotheses: 1) the microbial community in a P-limited soil will possess traits indicative of more abundant, diverse, and active mechanisms of P acquisition or mechanisms for the retention of organic matter substrates, and 2) greater soil respiration resulting from P amendment will be relatable to prior in-situ P limitation and to increased expression of genes involved in central anabolic and catabolic activities.

Section snippets

Materials and methods

Methods for soil physical and chemical measurements can be found in the supplemental material.

Relationships between environmental indices and CO2 respiration during incubation

Available soil phosphorus varied significantly among study locations (ANOVA, adj. p-value<0.05); it was lowest in Bisley ridge (BR) and El Verde valley (EV), intermediate in El Verde ridge (ER), and greatest in Bisley valley (BV) (Table 1). A positive correlation between available P and microbial biomass phosphorus (MBP) was observed in ridge soils (R2 = 0.82) (Fig. S3a). Positive correlations were also observed between MBP and microbial biomass carbon (MBC) (R2 = 0.77) and microbial biomass

Discussion

Phosphorus is often regarded as the primary nutrient limiting biological activities in tropical ecosystems (Vitousek, 1984; Tanner et al., 1998), yet many uncertainties remain concerning how P availability governs tropical soil microbial community composition and activities (Turner and Wright, 2014). In this study, P amendment (P+) of lab-incubated soils increased CO2 respiration by 14–23% for soils sampled from all but the site with the greatest total and bioavailable P; ER, BR, and EV were

Conclusions

In this study, community composition (determined with metagenomics) and activity assessments (including CO2 respiration, enzyme assays for organic carbon and phosphorus decomposition, and metatranscriptomics) together revealed how tropical soil microbiota respond acutely to an alleviation of P constraints. Functional signatures revealed community traits reflecting long-term adaptation to growth-restricting P conditions. This included the abundances of genes for α-glucosyl polymer biosynthesis,

Conflicts of interest

The authors declare no conflict of interest.

Acknowledgements

This research was sponsored by the Oak Ridge National Laboratory Directed Research and Development Program (to MAM); the ORNL ‘GO! Student Program’ (to ERJ); and the US National Science Foundation (award 1356288 to KTK). ORNL is managed by the University of Tennessee-Battelle, LLC, under contract DE-AC05-00OR22725 with the U.S. Department of Energy. We thank Grizelle Gonzalez of the US Forest Service International Institute of Tropical Forestry and Jess Zimmerman of the University of Puerto

References (67)

  • C.C. Cleveland et al.

    Nutrient additions to a tropical rain forest drive substantial soil carbon dioxide losses to the atmosphere

    Proceedings of the National Academy of Sciences

    (2006)
  • C.C. Cleveland et al.

    Phosphorus limitation of microbial processes in moist tropical forests: evidence from short-term laboratory incubations and field studies

    Ecosystems

    (2002)
  • C.C. Cleveland et al.

    Relationships among net primary productivity, nutrients and climate in tropical rain forest: a pan‐tropical analysis

    Ecology Letters

    (2011)
  • M.J.L. Coolen et al.

    The transcriptional response of microbial communities in thawing Alaskan permafrost soils

    Frontiers in Microbiology

    (2015)
  • M.P. Cox et al.

    SolexaQA: at-a-glance quality assessment of Illumina second-generation sequencing data

    BMC Bioinformatics

    (2010)
  • J.M. DeBruyn et al.

    Gemmatirosa kalamazoonesis gen. nov., sp. nov., a member of the rarely-cultivated bacterial phylum Gemmatimonadetes

    Journal of General and Applied Microbiology

    (2013)
  • R. Dephilippis et al.

    Glycogen and poly-β-hydroxybutyrate synthesis in Spirulina maxima

    Journal of General Microbiology

    (1992)
  • D. Derensy-Dron et al.

    β‐1, 3‐Galactosyl‐N‐acetylhexosamine phosphorylase from Bifidobacterium bifidum DSM 20082: characterization, partial purification and relation to mucin degradation

    Biotechnology and Applied Biochemistry

    (1999)
  • M. Drebot et al.

    Genetic assessment of stationary phase for cells of the yeast Saccharomyces cerevisiae

    Journal of Bacteriology

    (1990)
  • N. Fanin et al.

    Distinct Microbial Limitations in Litter and Underlying Soil Revealed by Carbon and Nutrient Fertilization in a Tropical Rainforest

    PLoS One

    (2012)
  • M. Givskov et al.

    Responses to nutrient starvation in Pseudomonas putida KT2442: two-dimensional electrophoretic analysis of starvation- and stress-induced proteins

    Journal of Bacteriology

    (1994)
  • S. Guindon et al.

    New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0

    Systematic Biology

    (2010)
  • K.J. Harrison et al.

    Gene Graphics: a genomic neighborhood data visualization web application

    Bioinformatics

    (2017)
  • J. Hultman et al.

    Multi-omics of permafrost, active layer and thermokarst bog soil microbiomes

    Nature

    (2015)
  • D. Hyatt et al.

    Prodigal: prokaryotic gene recognition and translation initiation site identification

    BMC Bioinformatics

    (2010)
  • N. Jaito et al.

    Characterization of a thermophilic 4-O-β-d-mannosyl-d-glucose phosphorylase from Rhodothermus marinus

    Bioscience Biotechnology & Biochemistry

    (2014)
  • E.R. Johnston et al.

    Metagenomics reveals pervasive bacterial populations and reduced community diversity across the Alaska tundra ecosystem

    Frontiers in Microbiology

    (2016)
  • K. Katoh et al.

    MAFFT multiple sequence alignment software version 7: improvements in performance and usability

    Molecular Biology and Evolution

    (2013)
  • E. Kopylova et al.

    SortMeRNA: fast and accurate filtering of ribosomal RNAs in metatranscriptomic data

    Bioinformatics

    (2012)
  • I. Letunic et al.

    Interactive tree of life (iTOL) v3: an online tool for the display and annotation of phylogenetic and other trees

    Nucleic Acids Research

    (2016)
  • S.H. Lillie et al.

    Reserve carbohydrate metabolism in Saccharomyces cerevisiae: responses to nutrient limitation

    Journal of Bacteriology

    (1980)
  • R. Mackelprang et al.

    Permafrost meta-omics and climate change

    Annual Review of Earth and Planetary Sciences

    (2016)
  • S.M. Mage et al.

    Parent material and topography determine soil phosphorus status in the Luquillo mountains of Puerto Rico

    Ecosystems

    (2013)
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    Current address: Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN, USA.

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