Brüggemann, N.

Louis, S.

Meier, R.

Schnitzler, J.P.

Steigner, D.

Zimmer, I.

English

Schnitzler, J. P.

KITopen

IMK-IFU Garmisch-PartenkirchenNicolas Brüggemann & Jörg-Peter Schnitzler | IMK-IFU | Garmisch1st workshop on aerobic methane formation, 26/27 Feb 2009, MainzKIT – a Cooperation betweenKarlsruhe Research Centerand University of KarlsruheAerobic methane formation in Grey poplar plants grown under sterile conditionsNicolas Brüggemann, Rudolf Meier, Dominik Steigner, Ina Zimmer, Sandrine Louis & Jörg-Peter SchnitzlerKarlsruhe Research CenterInstitute of Meteorology and Climate ResearchAtmospheric Environmental Research (IMK-IFU)Garmisch-PartenkirchenGermanyIMK-IFU Garmisch-PartenkirchenNicolas Brüggemann & Jörg-Peter Schnitzler | IMK-IFU | Garmisch1st workshop on aerobic methane formation, 26/27 Feb 2009, MainzKIT – a Cooperation betweenKarlsruhe Research Centerand University of KarlsruheIntroductionObjections to the experimental design of Keppler et al. (2006), criticizingthe use of static chambers and methane-free air: e.g.,Kirschbaum et al. (2006), Functional Plant Biology 33: 521–530Dueck et al. (2007), New Phytologist 175: 29–35No observation of aerobic methane emission from plants: e.g.,Dueck et al. (2007), New Phytologist 175: 29–35Beerling et al. (2008), Global Change Biology 14: 1821–1826Kirschbaum & Walcroft, Biogeosciences 5: 1551–1558Observation of aerobic methane emission from plants: e.g.,Vigano et al. (2008), Biogeosciences 5: 937–947Wang et al. (2008), Environmental Science & Technology 42: 62–68Mechanisms of aerobic methane formation: e.g.,Keppler et al. (2008), New Phytologist 178: 808–814McLeod et al. (2008), New Phytologist 180: 124–132Messenger et al. (2009), Plant, Cell & Environment 32: 1–9IMK-IFU Garmisch-PartenkirchenNicolas Brüggemann & Jörg-Peter Schnitzler | IMK-IFU | Garmisch1st workshop on aerobic methane formation, 26/27 Feb 2009, MainzKIT – a Cooperation betweenKarlsruhe Research Centerand University of KarlsruheOpen research questions• Missing proof for the absence of methanogenic microorganisms potentially contributing to aerobic methane emission from plants• Convincing evidence that aerobic methane originates in living plant materialIMK-IFU Garmisch-PartenkirchenNicolas Brüggemann & Jörg-Peter Schnitzler | IMK-IFU | Garmisch1st workshop on aerobic methane formation, 26/27 Feb 2009, MainzKIT – a Cooperation betweenKarlsruhe Research Centerand University of KarlsruheOur experimental design• Plant species: Grey poplar (Populus x canescens, syn. Populustremula x P. alba), derived from cell cultures under sterile conditions• Plants on sterile medium in gas-tight flasks in CH4-free air• Headspace was exchanged with synthetic air containing 20% of oxygen and 385 ppm 13CO2 (99 at% 13C)• Flasks were kept in glove box filled with pure N2 for 33 days under a 16/8 h light/dark regime • GC-IRMS analysis of methane in the headspace• Molecular biological analysis of plant material and medium for the methyl coenzyme M reductase alpha subunit (mcrA) gene • EA-IRMS of bulk plant material after end of the experimentIMK-IFU Garmisch-PartenkirchenNicolas Brüggemann & Jörg-Peter Schnitzler | IMK-IFU | Garmisch1st workshop on aerobic methane formation, 26/27 Feb 2009, MainzKIT – a Cooperation betweenKarlsruhe Research Centerand University of KarlsruhePlant materialWild type Populus × canescens (Aiton) Sm. (syn. Populus tremula × P. alba) lines,amplified by micro-propagationThe poplar plants were grown under standard conditions of 27°C : 24°C (day : night) and a light period of 16 h with approx. 100 μmol m−2s−1 photosynthetic photon flux density (PPFD)7-8 plantlets were transferred under sterile conditions to 1-l sterile glass flasks, containingsterilized quartz sand and MS mediumThe flasks were sealed with screw caps and sterilized valves; the inlet ports wereadditionally equipped with sterile filters (0.22 μm pore size)IMK-IFU Garmisch-PartenkirchenNicolas Brüggemann & Jörg-Peter Schnitzler | IMK-IFU | Garmisch1st workshop on aerobic methane formation, 26/27 Feb 2009, MainzKIT – a Cooperation betweenKarlsruhe Research Centerand University of KarlsruheSample inletSample volumeCO oxidationto CO2Condens tion of sample air with freezing of CO2To sample inletAir dryerTo Preconand IRMSCH4sampling flowchartForward primer: GGATTCACACARTAYGCWACAGCReverse primer: TCATBGCRTAGTTHGGRTAGT Databases: thousands of mcrA sequencesbut only „few“ arefull-length Alignment: to see conservedregions and design primersmrcA primers used for PCR IMK-IFU Garmisch-PartenkirchenNicolas Brüggemann & Jörg-Peter Schnitzler | IMK-IFU | Garmisch1st workshop on aerobic methane formation, 26/27 Feb 2009, MainzKIT – a Cooperation betweenKarlsruhe Research Centerand University of KarlsruheDays after 13CO2 addition0 7 33CH4 mixing ratio [ppbv]050100150200250 empty medium medium + plants aabababc abcbc bccdACH4 formationCH4 release rate from plants (mean, ± s.e.m.)0-7 days: 0.70 ± 0.37 ng g-1 dry weight h-17-33 days: 0.16 ± 0.11 ng g-1 dry weight h-10-33 days: 0.24 ± 0.06 ng g-1 dry weight h-1n = 5, ± SEIMK-IFU Garmisch-PartenkirchenNicolas Brüggemann & Jörg-Peter Schnitzler | IMK-IFU | Garmisch1st workshop on aerobic methane formation, 26/27 Feb 2009, MainzKIT – a Cooperation betweenKarlsruhe Research Centerand University of KarlsruheDays after 13CO2 addition0 7 33-40-30-20-10010203040 emptymedium medium & plants13 C of methane [‰ vs. V-PDB] cbaba a aa aaB13C of CH4IMK-IFU Garmisch-PartenkirchenNicolas Brüggemann & Jörg-Peter Schnitzler | IMK-IFU | Garmisch1st workshop on aerobic methane formation, 26/27 Feb 2009, MainzKIT – a Cooperation betweenKarlsruhe Research Centerand University of KarlsruheRelationship between 13C-CH4 and 13C of bulk plant material13C of bulk plant material [‰ vs. V-PDB]180 200 220 240 260 280 300 32013 C of CH4 [‰ vs. V-PDB]-40-20020406080100rr2 = 0.612P = 0.118y = 1.28 x - 300.59 IMK-IFU Garmisch-PartenkirchenNicolas Brüggemann & Jörg-Peter Schnitzler | IMK-IFU | Garmisch1st workshop on aerobic methane formation, 26/27 Feb 2009, MainzKIT – a Cooperation betweenKarlsruhe Research Centerand University of KarlsruheElectrophoretic analysis of PCR products of medium & plantsPcPSY PcTUB mcrAPcPSY = oligonucleitid primer specific for the Populus × canescens phytoene synthase genePcTUB = oligonucleitid primer specific for the Populus × canescens β-tubulin genemcrA = oligonucleitid primer specific for the methyl coenzyme M reductase alpha subunitIMK-IFU Garmisch-PartenkirchenNicolas Brüggemann & Jörg-Peter Schnitzler | IMK-IFU | Garmisch1st workshop on aerobic methane formation, 26/27 Feb 2009, MainzKIT – a Cooperation betweenKarlsruhe Research Centerand University of KarlsruheWang et al., 2008, nine emittingspecies (35 non-emitting species)0.5–13.5Vigano et al., 2008, for a fully 13C-labelled wheat leaf of Dueck et al., 2007, without UV light0.03Our work0.16–0.7Dueck et al., 2007, six species–10–42 (not significantly different from 0)Vigano et al., 2008, for a fully 13C-labelled wheat leaf of Dueck et al., 2007, without UV lightUp to 32Beerling et al., 2008, two species32–49 (not significantly different from 0)Keppler et al., 2006, five species12–370Kirschbaum & Walcroft, 2008; Nisbet et al., 2009, two species;NDReferencesng CH4 g−1 DW h−1Range of aerobic CH4 from living and detached plant materialIMK-IFU Garmisch-PartenkirchenNicolas Brüggemann & Jörg-Peter Schnitzler | IMK-IFU | Garmisch1st workshop on aerobic methane formation, 26/27 Feb 2009, MainzKIT – a Cooperation betweenKarlsruhe Research Centerand University of KarlsruheSummary• We have observed release of 13C-labelled CH4 from poplarsignificantly different from zero under low (UV-free) light conditionsafter 13CO2 labelling• Our work is the first molecular biological proof for the absence of methanogenic microorganisms in plants emitting CH4 under aerobicconditions• However, poplar methane emission rates are at the lower end of thereported CH4 emission rates from living or detached plant material• The 13C-label was detectable in CH4 released from the plantsalready several minutes after start of 13CO2 labellingIMK-IFU Garmisch-PartenkirchenNicolas Brüggemann & Jörg-Peter Schnitzler | IMK-IFU | Garmisch1st workshop on aerobic methane formation, 26/27 Feb 2009, MainzKIT – a Cooperation betweenKarlsruhe Research Centerand University of KarlsruheThe “perfect” aerobic methane experiment?• Experiments at ambient gas (CH4, O2, CO2) concentration levels• Stable isotope labelling essential to differentiate between plant and  atmospheric methane• Analysis of plant-internal reactive oxygen species (ROS)• Molecular biological verification of the absence of methanogenes• Application of defined stress situations initiating ROS formation• …(open for discussion)Goal: Elucidation of CH4 mechanism(s) with simultaneous determination of realistic emission rates

Aerobic methane formation in Grey poplar plants grown under sterile conditions

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Aerobic methane formation in Grey poplar plants grown under sterile conditions

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