BACKGROUND AND AIMS: Rootless carnivorous plants of the genus Utricularia are important components of many standing waters worldwide, as well as suitable model organisms for studying plant-microbe interactions. In this study, an investigation was made of the importance of microbial dinitrogen (N2) fixation in the N acquisition of four aquatic Utricularia species and another aquatic carnivorous plant, Aldrovanda vesiculosa. METHODS: 16S rRNA amplicon sequencing was used to assess the presence of micro-organisms with known ability to fix N2. Next-generation sequencing provided information on the expression of N2 fixation-associated genes. N2 fixation rates were measured following (15)N2-labelling and were used to calculate the plant assimilation rate of microbially fixed N2. KEY RESULTS: Utricularia traps were confirmed as primary sites of N2 fixation, with up to 16 % of the plant-associated microbial community consisting of bacteria capable of fixing N2. Of these, rhizobia were the most abundant group. Nitrogen fixation rates increased with increasing shoot age, but never exceeded 1·3 μmol N g(-1) d. mass d(-1). Plant assimilation rates of fixed N2 were detectable and significant, but this fraction formed less than 1 % of daily plant N gain. Although trap fluid provides conditions favourable for microbial N2 fixation, levels of nif gene transcription comprised <0·01 % of the total prokaryotic transcripts. CONCLUSIONS: It is hypothesized that the reason for limited N2 fixation in aquatic Utricularia, despite the large potential capacity, is the high concentration of NH4-N (2·0-4·3 mg L(-1)) in the trap fluid. Resulting from fast turnover of organic detritus, it probably inhibits N2 fixation in most of the microorganisms present. Nitrogen fixation is not expected to contribute significantly to N nutrition of aquatic carnivorous plants under their typical growth conditions; however, on an annual basis the plant-microbe system can supply nitrogen in the order of hundreds of mg m(-2) into the nutrient-limited littoral zone, where it may thus represent an important N source.

Department of Ecosystem Biology Department of Experimental Plant Biology Faculty of Science University of South Bohemia Branišovská 31 CZ 37005 České Budějovice Czech Republic

Department of Ecosystem Biology Department of Experimental Plant Biology Faculty of Science University of South Bohemia Branišovská 31 CZ 37005 České Budějovice Czech Republic Institute of Hydrobiology Biology Centre AS CR Na Sádkách 7 CZ 37005 České Budějovice Czech Republic

Department of Ecosystem Biology Department of Experimental Plant Biology Faculty of Science University of South Bohemia Branišovská 31 CZ 37005 České Budějovice Czech Republic Institute of Plant Molecular Biology Biology Centre AS CR Branišovská 31 CZ 37005 České Budějovice Czech Republic

Department of Grassland Study Technical University Munich Alte Akademie 12 Freising Weihenstephan Germany

Institute of Applied Informatics Faculty of Science University of South Bohemia Branišovská 31 CZ 37005 České Budějovice Czech Republic

Institute of Experimental Botany AS CR Rozvojová 263 CZ 16502 Prague 6 Lysolaje Czech Republic

Institute of Genomics and Systems Biology Argonne National Laboratory Argonne and Computation Institute University of Chicago Chicago IL USA

Institute of Hydrobiology Biology Centre AS CR Na Sádkách 7 CZ 37005 České Budějovice Czech Republic

Section of Plant Ecology Institute of Botany AS CR Dukelská 135 CZ 37982 Třeboň Czech Republic

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