Guber, A. K.

Koestel, Johannes

Kravchenko, A. N.

Kuzyakov, Y.

Quigley, M. Y.

Razavi, B. S.

Robertson, G. P.

English

Epsilon Open Archive

MATTERS ARISINGReply to: “Variables in the effect of land use on soilextrapore enzymatic activity and carbonstabilization” by Glenn (2020)A. N. Kravchenko 1,2,3✉, A. K. Guber1,2, B. S. Razavi4, J. Koestel5, M. Y. Quigley 1, G. P. Robertson 1,2,6 &Y. Kuzyakov3,7,8REPLYING TO Glenn, M. Nature Communications https://doi.org/10.1038/s41467-020-19900-9 (2020)We appreciate the comments by Glenn1 regarding ourstudy of the plant system effects on soil pore char-acteristics and their implications for soil carbon sto-rage (Kravchenko et al.2). We value the opportunity to respondand are happy to clarify the nature of the presented data and thedesign of our study, lack of clarity in which apparently generatedGlenn’s1 concerns. We also would like to further clarify themechanisms that underline the observed associations betweenpore sizes and enzyme activities, which were not articulated indetail in the original paper.The first concern of Dr. Glenn is related to the data presented inFig. 4a of the original paper. The presented results are not theabsolute values of the enzyme activities, but relative activitiesstandardized across each zymography map within each soil core.By construction, the average activity across each individual com-plete zymography map was set equal to zero. Hence, the averagesfor all systems across all pore sizes are also zeros, making anygeneral statement regarding which system has higher/lowerenzyme activity based on Fig. 4a data meaningless. The standar-dization was necessary to explore the distribution of enzymeactivities associated with pores of specific sizes. The only mean-ingful comparisons using this data are the comparisons among thepore size groups within individual systems and this is what isreported in the study. From that perspective, the low values meanthat the enzyme activities for particular pore size groups wereclose to the overall averages. Positive values indicated the pre-valence of the above average values associated with certain poresize groups and negatives—below average values.Association of the above-average enzyme activities with largepores in switchgrass but not in biodiverse systems is what formedthe basis for our main conclusions. It is not the overall differencesin microbial activities (those were similarly high in switchgrassand biodiverse systems (Fig. 1a in the original paper)), and notthe overall differences in the enzyme activities (Suppl Fig. 4 in theoriginal paper), but the fascinating differences in spatial patternsof the enzyme activities is what led to our conclusions.Our experimental results are consistent with mechanisticunderstanding of the role that pores of various sizes play in soilfunctioning. While it is discussed in the original manuscript,here we further elucidate the relationships (Fig. 1). The finest pores(Ø < 1 µm) only minimally contribute to microbial activitiesbecause they are inaccessible to soil microorganisms due to sizelimitations. They experience only negligible influx of extracellularenzymes due to lack of diffusion of large enzyme molecules3. Thewater contained in the finest pores, while plentiful, is unavailabledue to high matric potentials; and their O2 supply is limited(Fig. 1). However, these pores are excellent for storage of organicmolecules enabling entombing and long-term carbon sequestrationby spatial exclusion4. While pores in 1–10 µm Ø size range canharbor individual microorganisms, they do not provide enoughspace for microbial colonies5, not surprisingly having low micro-bial activity. Microbial colonies, crucial for C and N cycling, can bebuilt within the pores with diameters at least a few tens of microns.The same pores are occupied by young fine roots which generatemaximal rhizodeposition. The water supply in these pores isoptimal and O2 is unlimited. The pores a few hundreds of micronsin diameter are well accessible to soil animals grazing on bacteriaand fungi. Moreover, these pores tend to drain and dry out thefastest, shortening the periods optimal for microbial activity.Tendencies for higher enzyme activities in 30–150 µm pores insoils of low diversity systems observed in our study agree with thenotion of pores of this size range forming an optimal habitat formicrobial colonies, with plentiful nutrient supply via rhizodepo-sition and water influxes, optimal balance between air and waterhttps://doi.org/10.1038/s41467-020-19901-8 OPEN1 Department of Plant, Soil and Microbial Sciences, Michigan State University, East Lansing, MI 48824, USA. 2DOE Great Lakes Bioenergy Research Center,Michigan State University, East Lansing, MI, USA. 3 Department of Agricultural Soil Science, University of Göttingen, Göttingen, Germany. 4 Institute of plantnutrition and soil science, Kiel University, Kiel, Germany. 5 Swedish University of Agricultural Sciences, Uppsala, Sweden. 6W. K. Kellogg Biological Station,Michigan State University, Hickory Corners, MI 49060, USA. 7 Institute of Physicochemical and Biological Problems in Soil Science, Pushchino 142290,Russia. 8 RUDN University, Moscow, Russia. ✉email: kravche1@msu.eduNATURE COMMUNICATIONS |         (2020) 11:6427 | https://doi.org/10.1038/s41467-020-19901-8 | www.nature.com/naturecommunications 11234567890():,;regimes, and low predation. Consequently, they experience highmicrobial densities and, hence, high enzyme activities. Last butnot least, very fine roots populating these pores are anotherimportant source of extracellular enzyme release.In the diverse plant systems, high abundance, connectivity, andwide spread of such pores through the soil matrix led to ubiquitouspresence of microorganisms, fine roots, and enzymes. While in thelow diversity systems, where such pores were not so prevalent andsparsely placed, their positive contribution to microbial activity andresultant enzyme patterns was clearly pronounced.The second concern of Dr. Glenn is that the observed differ-ences in soil properties cannot be attributed to plant diversity butare generated by other sources, such as differences in inherent soilcharacteristics, faunal and groundwater movements, and histor-ical management differences. As described in the original paper,the experimental design of our field study is a randomizedcomplete block with five replications. At the study initiation in2007, the blocks were laid out to minimize spatial variabilitypatterns in inherent soil and topographical characteristics, andthe plant system treatments were randomly assigned to theexperimental plots within each block. Thus, it can be safelyconcluded that the developed differences in the soil characteristicswere caused by the plant system effects, of which plant diversity isthe most pronounced feature. In fact, the only explanatory vari-able which can be used to generate cause-and-effect conclusionsin this study is plant system/plant diversity. The influence of theother inherent factors (mineral content, depth of surface andsubsurface horizons, historical management practices) can beconsidered as equally affecting all studied plant systems, due tothe replicated blocked experimental design with random treat-ment assignment.We feel strongly that it is not enough to stop at the conclusionthat plant diversity causes differences in soil pore characteristics.Plant diversity manifests its influence on soil pore structurethrough a variety of physical, chemical, and biological controls. Itinfluences soil organic matter levels, quality of soil carbon inputs,macro and mesofauna and their movements. Those factors weremost likely among the key agents through which differences inplant diversity created the differences observed here in soil poresize distributions and enzyme localization patterns between plantsystems. It is of outmost importance to focus future research effortson determining drivers operating in diverse plant communitiesthat shape soil pore architecture. While we can speculate onpotential contributions of uncontrolled variables, the only scienti-fically warranted conclusion from our study is that the plant sys-tems with different diversity levels caused the observed differences.Reporting summary. Further information on research design is available in the NatureResearch Reporting Summary linked to this article.Data availabilityNo new data were generated for this response. Data collected for the original study byKravchenko et al.2 are provided as a Source Data file and will be preserved in the KBSLTER database available at http://lter.kbs.msu.edu/datatables.Fig. 1 Intra-pore environmental conditions related to biological and enzyme activities as a function of pore size. Water and gas fluxes are very slow infew-micron-sized pores, increasing rapidly with increasing pore size (blue lines and text). Pores <1 µm diameter are too small for hosting microorganisms,and only single bacteria cells or fungal hyphae can be hosted within <10 µm pores. Pores with 10 to few-hundred-µm diameters accommodate fine plantroots and root hairs and are the locations of maximal rhizodeposition. This pore range is optimal for microorganisms due to plant inputs, and optimal waterand O2 availabilities (top graph). This is the range of pores with the highest enzyme activities elucidated by zymography and µCT2 results of our study(shaded green). The pores of larger size are occupied by predators decreasing microbial density by grazing. Carbon sequestration is mainly taking place inthe pores smaller than few micrometers by entombing: spatial exclusion from microbial and enzymatic activities (shaded red).MATTERS ARISING NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-19901-82 NATURE COMMUNICATIONS |         (2020) 11:6427 | https://doi.org/10.1038/s41467-020-19901-8 | www.nature.com/naturecommunicationsCode availabilityNo new codes were generated for this response. Data and image analyses codes for theoriginal study by Kravchenko et al.2 are available from the authors upon request.Received: 10 November 2019; Accepted: 2 November 2020;References1. Glenn, M. Matters arising: Variables in the effect of crop field biodiversity onsoil extrapore enzymatic activity and carbon stabilization. Nat. Commun. Inpress. https://doi.org/10.1038/s41467-020-19900-9 (2020).2. Kravchenko, A. N. et al. Microbial spatial footprint as a driver of soil carbonstabilization. Nat. Commun. 10, 3121 (2019).3. Guber, A. et al. Quantitative soil zymography: mechanisms, processes ofsubstrate and enzyme diffusion in porous media. Soil Biol. Biochem. 127,156–167 (2018).4. Liang, C., Schimel, J. P. & Jastrow, J. D. The importance of anabolism inmicrobial control over soil carbon storage. Nat. Microbiol. 2, 17105 (2017).5. Kuzyakov, Y. & Blagodatskaya, E. Microbial hotspots and hot moments in soil:concept & review. Soil Biol. Biochem. 83, 184–199 (2015).AcknowledgementsWe would like to thank Chelsea Mamott of GLBRC communication team for the artworkand figure preparation.Author contributionsA.N.K. and Y.K. devised the responses and wrote the manuscript. A.K.G., B.S.R., J.K.,M.Y.Q., and G.P.R. contributed to manuscript writing and reviewed the manuscript.Competing interestsThe authors declare no competing interests.Additional informationSupplementary information is available for this paper at https://doi.org/10.1038/s41467-020-19901-8.Correspondence and requests for materials should be addressed to A.N.K.Peer review information Nature Communications thanks Aimeric Blaud for theircontribution to the peer review of this work.Reprints and permission information is available at http://www.nature.com/reprintsPublisher’s note Springer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations.Open Access This article is licensed under a Creative CommonsAttribution 4.0 International License, which permits use, sharing,adaptation, distribution and reproduction in any medium or format, as long as you giveappropriate credit to the original author(s) and the source, provide a link to the CreativeCommons license, and indicate if changes were made. The images or other third partymaterial in this article are included in the article’s Creative Commons license, unlessindicated otherwise in a credit line to the material. If material is not included in thearticle’s Creative Commons license and your intended use is not permitted by statutoryregulation or exceeds the permitted use, you will need to obtain permission directly fromthe copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.© The Author(s) 2020NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-19901-8 MATTERS ARISINGNATURE COMMUNICATIONS |         (2020) 11:6427 | https://doi.org/10.1038/s41467-020-19901-8 | www.nature.com/naturecommunications 3

Reply to: "Variables in the effect of land use on soil extrapore enzymatic activity and carbon stabilization" by Glenn (2020)

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Reply to: "Variables in the effect of land use on soil extrapore enzymatic activity and carbon stabilization" by Glenn (2020)

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