07 Oct 2014
Supporting Information
Colonization of rice roots with methanogenic archaea controls
photosynthesis-derived CH4 emission
Judith Pump, Jennifer Pratscher, Ralf Conrad
Experimental
Pulse-labeling experiments
Pulse-labeling experiments were performed on rice-planted microcosms as described by
Pump and Conrad (2014). The microcosms were prepared in plastic pots (1 l volume), which
were filled with 0.8 kg air-dried, mechanically ground and sieved (< 2 mm) Italian paddy soil.
Alternatively, 80 g sieved soil was amended with 720 g of a mixture (80:1) of quartz sand
(Roth, Karlsruhe, Germany) and vermiculite (Thinex New Media, Dortmund, Germany). The
pots were flooded with demineralized water, and after one week, each soil microcosm was
planted with one fungicide-treated, surface-sterilized and pre-germinated rice seedling (Oryza
sativa, var. Koral, type japonica). The planted microcosms were fertilized every second week
with half strength Hoagland’s solution (Hoagland and Arnon, 1950) and cultivated in the
greenhouse at 25°C, ~ 75% humidity and a photoperiod of 12 h. Demineralized water was
added daily to maintain waterlogged conditions.
Pulse-labeling experiments were performed at different growth stages of the plants, the
early vegetative (2-3 weeks), late vegetative (6-7 weeks), reproductive (9-10 weeks) and
ripening (13-14 weeks) stage using duplicate or triplicate microcosms, which were placed in a
climate growth cabinet (Conviron, Berlin, Germany) under controlled conditions (25°C, ~ 35
kLux light intensity, photoperiod of 12 h). The microcosms were covered with an acrylic
chamber (18 l volume) and pulse-labeled by injection of 170 ml 13CO2 (99 atom% 13C,
Sigma-Aldrich, Taufkirchen, Germany) giving an initial CO2 concentration in the headspace
of about 1%. Gas samples (1 ml) were taken, and CH4 and CO2 concentrations were analyzed
by gas chromatography. The isotopic composition (13C/12C) of CH4 was analyzed using a preconcentration gas chromatograph combustion isotope ratio mass spectrometer system
(PreCon-GC-C-IRMS) as described by Brand (1996) and Rice et al. (2001). After 102 h,
microcosms were destructively sampled.
DNA extraction and molecular analyses
The roots from the destructively sampled microcosms were shaken to remove large soil
aggregates and loosely adhering soil. The remaining attached soil was sampled with a sterile
spatula and used for DNA extraction. The roots were carefully washed, first with tap water
and then with sterile demineralized water. Soil and roots were immediately shock-frozen in
liquid nitrogen and stored at -80°C until analysis. DNA was extracted from soil or roots using
the FastDNA SPIN Kit for Soil (MP Biomedicals) following the manufacturer’s instructions
in combination with cell lyses via bead beating (FastPrep-24 Instrument; MP Biomedicals)
and chemical removal of humic acids by washing with sterile 5.5 M guanidine thiocyanate.
DNA yield and purity were measured using a NanoDrop 1000 Spectrophotometer (Thermo
Fisher Scientific, Schwerte, Germany). Extracted DNA was stored at -20°C until further
processing.
Gene copies of archaeal 16S rRNA and mcrA were quantified in duplicate by qPCR
using an iCycler iQ thermocycler (Bio-Rad) and the thermal program modified from Lueders
et al. (2004). The qPCR conditions targeting archaeal 16S rRNA genes included SYBR Green
JumpStart Taq Ready Mix containing 1.25 U Taq DNA polymerase and 0.2 mM dNTP, 3 mM
MgCl2 (Sigma-Aldrich), 0.8 µg µl-1 BSA (Roche), 1:1,000 Fluorescein Calibration Dye (BioRad Laboratories GmbH, Munich, Germany), 0.66 µM of each primer 364F (Burggraf et al.,
1997) and 934b (Grosskopf et al., 1998a). Similarly, mcrA genes were quantified, but using
3.5 mM MgCl2 and 0.25 µM of each primer mlas and mcrA-rev (Steinberg and Regan, 2008).
Archaeal 16S rRNA and mcrA gene amplicons of Methanosarcina barkeri with known copy
numbers of the respective target gene were used as qPCR standards in duplicate dilution
series for construction of calibration curves in each reaction.
For T-RFLP analyses, partial archaeal 16S rRNA genes were amplified using the
forward primer A109f and the reverse primer A934b, which was fluorescently-labeled with
FAM (6-carboxyfluorescein) at the 5’-terminus (Grosskopf et al., 1998b). Methanogenspecific mcrA genes were amplified using the FAM-labeled forward primer mlas and the
reverse primer mcrA-rev (Steinberg and Regan, 2008). 16S rRNA and mcrA gene amplicons
were purified with the MinElute PCR Purification Kit (Qiagen GmbH, Hilden, Germany),
digested with TaqI and FastDigest Sau96I (Fermentas), respectively, and post-cleaned using
SigmaSpin Post-Reaction Purification Columns (Sigma-Aldrich) following the manufacturer's
instructions. Cleaned, digested amplicons (2 µl) were mixed with 11 µl of HiDi formamide
(Applied Biosystems, Darmstadt, Germany) and 0.3 µl of MapMarker 1000 (BioVentures,
Murfreesboro, USA). After denaturation at 95°C for 3 min, fluorescently labeled terminal
restriction fragments (T-RFs) were size separated on an ABI 3130 automated sequencer
(Applied Biosystems). T-RFLP electropherograms were analyzed using GeneMapper
Software Version 4.0 (Applied Biosystems) by peak area integration of each single T-RF. TRFLP profiles were processed as described by Lueders and Friedrich (2003).
Clone libraries were constructed after PCR amplification of archaeal 16S rRNA and
mcrA genes using primers without FAM-label. The purified gene amplicons were cloned
using the pGEM-T Vector System cloning kit (Promega). Randomly selected clones were
commercially sequenced from both ends (GATC Biotech AG, Konstanz, Germany) using the
primers T7f and M13r targeting flanking regions of the insert. Clone sequences were imported
into the ARB software package (Ludwig et al., 2004) for quality check, alignment and
phylogenetic tree construction. Phylogenetic analysis of the sequences were done using the
SILVA 108 reference dataset for archaeal 16S rRNA and a custom generated reference
dataset with reference sequences from NCBI for mcrA sequences. All clone sequences from
this study were deposited at GenBank (http://www.ncbi.nlm.nih.gov) under the accession
numbers KM273476 - KM273781 and KM273264 - KM273475 (16S rRNA and mcrA,
respectively).
T-RFs were assigned to phylogenetic groups by using the restriction sites detected in the
clone sequences (Table 1), by using assignments described in the literature (Chin et al., 1999;
Chin et al., 2004; Kemnitz et al., 2004; Liu et al., 2012; Peng et al., 2008), and by screening
mcrA public data bases for Sau96I restriction sites. T-RFs with 465 bp length have no
restriction site and thus, cannot be assigned unambiguously.
Reference List
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Table S1. Assignment of terminal restriction fragments (T-RFs) to phylogenetic groups based
on 266 archaeal 16S rRNA gene and 193 mcrA sequences.
Marker genes
Phylogenetic affiliation
Terminal restriction
fragment length (bp)
Number
of clones
Archaeal 16S rRNA
Crenarchaeota
> 700
32
Nitrososphaera (Thaumarchaeota)
737, 186
89
Methanocellales
391
35
Methanosaetaceae
282
47
Methanosarcinaceae
491, 184
50
Methanobacteriaceae
87, 89, 91
13
Methanosarcinaceae
290, 357, 390, 457
59
Methanobacteriales
369, 372, 434, 438, (465)
38
Methanosaetaceae
110, 220, 384
61
Methanocellales
201, 403
35
mcrA