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Who are alpine bistort’s ( vivipara L.) playmates? Metaproteomics reveals the key microorganisms in the rhizosphere of a widespread arctic- alpine

Conference Paper · October 2013

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Iva Pitelkova Radovan Hynek Tromsø Museum University of Chemistry and Technology, Prague

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Sunil Mundra Pernille Bronken Eidesen United Arab Emirates University University Centre in Svalbard (UNIS)

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The user has requested enhancement of the downloaded file. Who are alpine bistort’s (Bistorta vivipara L.) playmates? Metaproteomics reveals the key microorganisms in the rhizosphere of a widespread arctic-alpine plant

Iva Hlavackova, Jiri Santrucek, Radovan Hynek Sunil Mundra, Pernille Bronken Eidesen Institute of Chemical Technology, Prague The University Centre in Svalbard, Longyearbyen

Introduction Biodiversity based on identified proteins

Plants provide microorganisms with various growth substrates and phys- iological habitats. The "hot spot" of plant-microbes interactions is a root In average, we identified approximately 160 proteins in each sample (bird cliff Sample H D ACE surface and zone around roots influenced directly by plant - rhizosphere. In in spring and summer, coal mine in summer, control site in summer) and we Bird cliff (spring) 2.4 7.9 25.3 a mycorrhizal association, the fungus colonizes the host plant’s roots, either detected approximately 80 species of microorganisms at each site based on Bird cliff (summer) 2.5 8.2 33.6 intracellularly as in arbuscular mycorrhizal fungi or extracellularly as in phylogenetic origin of proteins. Spring sampling at bird cliff showed lower Coal mine (summer) 2.5 8.3 36.9 ectomycorrhizal fungi. Mycorrhizal symbiosis is of crucial ecological role since biodiversity comparing to the summer sample (similarly as in Muhlmann Control (summer) 2.5 8.3 38.7 mycorrhizae influence plant growth and nutrient uptake. Mycorrhizal et al. 2008), the highest species richness was detected in summer at control H/D - Shannon’s/Simpson’s diversity index, ACE - Abundance Coverage richness Estimator are often more resistant to diseases and are also more resistant to the effects site Isdamen. of drought.

Alpine bistort (Bistorta vivipara L.) is one of few herbaceous species from the family which form both ectomycorrhizal and arbuscular mycorrhizal relationships. Based on DNA bar- coding, it has also been shown to have a specific Microorganisms detected in Alpine bistort’s rhizosphere root-associated bacterial community.

This arctic-alpine plant is widespread and displays remarkably large ecological amplitude; it is one of the first plant species colonising primary successional sites, but it also grows in closed vegetation. Alpine bistort is of great ecological importance as key food item for e.g. geese and ptarmigans in high Arctic. The succes of this species can be related to its ability to associate with soil microorganisms.

Our main goal is to document biodiversity Scientific question in living microbial community associated with Alpine bistort’s roots and its variability, both seasonal and due to environmental factors impact. Our Independently of the sampling site and season, the majority of the spectra were assigned to four major phylogenetic

future findings might explain why Alpin bistort is succesful in harsh envi- View publication stats groups: Bacteria (59-70 %), Viridiplantae (non-degraded plant proteins whose amount differed between spring and ronments and bring new understanding of biogeochemical processes in per- mafrost. summer samples, 7-16 %), Fungi (7-10 %) and Metazoa (10-13 %). A more detailed itemization of the bacterial community indicated a dominance of Proteobacteria (averaged 62 %), followed by Actinobacteria (8 % in spring sample and largely increased in summer samples to 20 % in average); the last Experimental design and sites description notable group was formed by Firmicutes (9 % in average). We observed seasonal changes in Proteobacteria community structure where β-Proteobacteria were less frequent in spring than in summer and -Proteobacteria were detected in In order to study the seasonal changes summer sample only. and the impact of environmental factors on community structure associated with In fungal lineages the major group was Ascomycota; the frequency of organisms from this group seems to be site specific roots of Alpine bistort, three sites near with the lowest abundance at coal mine (45 %) and the highest abundance at control site (80 %). The second largest Longyearbyen (Svalbard) were chosen with group was Basidiomycota whose low presence was observed at control site (20 %) and the highest number was observed contrasting characteristics; the sampling at coal mine (55 %). Previously, basidiomycetes were reported as main symbionts of Alpine bistort (Muhlmann et al. was repeated approximately every two 2008). months.

• Bird cliff rich in nitrogen supply (Bjørndalen, N78 ◦14:008 E15 ◦19:848) • Coal mine with extremely low soil pH (Mine 3, N78 ◦13:326 E15 ◦19:446) • Non affected control site (Isdamen, N78 ◦12:769 E15 ◦42:231) Biological processes in communities of microorganisms

39 75 36 Bird cliff Mine Control In contrast to DNA and RNA, 39 Functional classification of proteins identified in rhizosphere 50 Metaproteomic approach 19 of Alpine bistort showing a comparison between different 18 most proteins have actual metabolic function and environmental proteomics 19 sites in summer (bird cliff, coal mine, control site). In total, allows us to link the abundance and activity of these "working horses" in the 25 Numberof proteins

% of proteins% approximately 110 proteins from each site were assigned to 0 system to their phylogenetic origin. The detailed study of a given habitat 0 0 one or more of 12 categories selected for classification using 0 combining the identification of microbial proteins, their temporal distribu- Web Gene Ontology Annotation Plotting (WEGO) application.

tion and abundance together with the analysis of their phylogenetic origin transport is expected to provide substantially new insights into the role of microbial We observed only small differences in biological processes de- gene expression catabolic process diversity in biogeochemical processes. metabolic process response to stress biosynthetic process tected at different sites. The noteworthy observing is higher

protein metabolic process number of proteins related to energy production together with

phosphorus metabolic process higher number of stress related proteins and proteins involved carbohydrate metabolic process

Methods macromolecule biosynthetic process in phosphorous metabolism observed at coal mine. This find- ing suggest that microbial community at coal mine might be

nucleo-base, -side, -tide and nucleic acid m. p. stressed by lower availability of phosphorus caused by low pH. Soil from Alpine bistort’s rhizosphere was collected in June (early spring) generation of precursor metabolites and energy and in July (summer) at all three sites where four plots where established with randomised design. To detect microorganisms present in rhizosphere, proteins were extracted from soil using phenol (Keiblinger et al. 2012). Next, protein samples were subjected to SDS-PAGE in order to clean samples from interfering substances (e. g. humic acids) and to decrease samples complexity. Environmental proteomics proved to be useful tool for studying microbial community Gel lines were cut to 7 pieces and trypsin digested. Proteins were identified Take home message ) and seems to be affected at some levels by environmental factors using liquid chromatography coupled with tandem mass spectrometry. In last structure associated with Alpine bistort’sProteobacteria roots. The community structure differs throughout the seasons, e. g. lineages step, identified proteins were assigned to phylogenetic and functional groups. occurence of the dominatingAscomycota phylum). (e. g. abundance of

References

Acknowledgement • Keiblinger K. M., Wilhartitz I. C., Schneider T. et al.: Soil metaproteomics - Comparative evaluation of protein extraction protocols. Soil Biol. Biochem. (2012) 54: 14–24. This research was supported by Ministry of Education, grant no. MSM • Muhlmann O., Bacher M. and Peintner U.: viviparum mycobionts on an alpine primary successional glacier forefront. Mycorrhiza 6046137305. (2008) 18: 87–95.