Characterization of Leaf Blade- and Leaf Sheath-Associated Bacterial Communities and Assessment of Their Responses to Environmental Changes in CO2, Temperature, and Nitrogen Levels under Field Conditions

Rice shoot-associated bacterial communities at the panicle initiation stage were characterized and their responses to elevated surface water-soil temperature (ET), low nitrogen (LN), and free-air CO2 enrichment (FACE) were assessed by clone library analyses of the 16S rRNA gene. Principal coordinate analyses combining all sequence data for leaf blade- and leaf sheath-associated bacteria revealed that each bacterial community had a distinct structure, as supported by PC1 (61.5%), that was mainly attributed to the high abundance of Planctomycetes in leaf sheaths. Our results also indicated that the community structures of leaf blade-associated bacteria were more sensitive than those of leaf sheath-associated bacteria to the environmental factors examined. Among these environmental factors, LN strongly affected the community structures of leaf blade-associated bacteria by increasing the relative abundance of Bacilli. The most significant effect of FACE was also observed on leaf blade-associated bacteria under the LN condition, which was explained by decreases and increases in Agrobacterium and Pantoea, respectively. The community structures of leaf blade-associated bacteria under the combination of FACE and ET were more similar to those of the control than to those under ET or FACE. Thus, the combined effects of environmental factors need to be considered in order to realistically assess the effects of environmental changes on microbial community structures.

each sample (6 mg) was suspended in 300 µL of MeOD/HEPES-d 18 buffer, which was prepared as described previously 1,2 . The mixture was heated at 50°C for 5 min while shaking at 1400 rpm in a Thermomixer comfort (Eppendorf AG, Hamburg, Germany) and then centrifuged (21,500 g, 5 min). The supernatant was used for the NMR experiments.

NMR spectroscopy
Sample solutions were transferred into 3.0 mm O.D.  103.5 mm NMR tubes (Norell, Landisville, NJ). NMR spectra were recorded on an Avance-500 spectrometer (Bruker BioSpin, Karlsruhe , Germany) equipped with a CryoProbe for 5-mm sample diameters operating at 500.23 MHz for 1 H and 125.80 MHz for 13 C. The temperature of all NMR samples was maintained at 298 K. The chemical shifts were referenced to the TMS group of sodium 2,2-dimethyl-2-silapentane-5-sulfonate (DSS, 0.5 mM in MeOD/HEPES-d 18 ) internal standard. 1 H NMR spectra were collected using the Bruker pulse program zgpr, and the following acquisition parameters were used: spectral width, 13 ppm; acquisition mode, sequential quadrature detection; offset frequency, 4.9 ppm; the proton 90° pulse, 16.3 s and 15.0 µs for carbon; relaxation delay, 10 s; number of scans, 128. 1 H 13 C heteronuclear quantum coherence (HSQC) spectra were collected using echo/antiecho gradient selection (the hsqcetgpsisp pulse program in the Bruker library) with the following parameters: 90° pulse values, 16.3 µs for proton; relaxation delay, 2 s; spectral width, 130 ppm (f1) and 10 ppm (f2); data points, 256 increments of 2048; scans, 64. The chemical shifts were referenced to the methyl group of a DSS internal standard (0.00 ppm for 1 H and 13 C).

Data analysis
The 1 H NMR spectra were processed using the TopSpin software (ver. 3.2, Bruker BioSpin). For principal component analysis (PCA), datasets were generated by subdividing spectra (10.000.50 ppm) into integrated regions of 0.04 ppm each using the Amix software (ver. 3.9.14, Bruker BioSpin). The integrated data were then normalised to total intensity of all the variables. The residual CH 2 DOD and DSS signals were excluded before further analysis. PCA were performed using the SIMCA software (ver. 13.0.3.0, Umetrics, Umeå, Sweden), and Pareto scaling was applied to PCA.

Annotation of signals
The 1 H 13 C HSQC spectra were processed with NMRPipe and analyzed using NMRDraw 3 . Metabolite signals were annotated using a semi-automated annotation program, SpinAssign (http://prime.psc.riken.jp/). Candidate metabolites for each peak were selected from standard compounds by comparing the chemical shift difference. A compound was selected when the chemical shift difference between the standard and queried peak was less than 0.03 ppm and 0.53 ppm for 1 H and 13 C, respectively.

Supplementary results
PCA was carried out using the datasets containing all 224 variables from the 1 H NMR spectra. Score and loading plots of leaf blade and sheath are shown in Figure S1. The score plot reflects the relative proportion of metabolites since each variable was scaled to the total intensity of the corresponding spectrum. The first and second principle components (PC1 and PC2) describes the effect of CO 2 concentration and difference in organ, respectively. The variation on PC1 was explained primarily on the differential abundance of sucrose (Suc) and glycerolipids (GLs) between ambient and FACE condition; leaves grown under FACE condition were characterised by higher levels of Suc, and lower amounts of GLs. Comparison of the raw spectra, that were normalized to the intensity of DSS also confirmed such tendency of increasing and decreasing amounts of Suc and GLs, respectively (data not shown). The variation along PC2 was due to differences in the relative proportion of Suc, fructose (Fru), glucose (Glc), and formate. The raw spectra showed that the total metabolite concentration was lower in leaf sheath than leaf blade, and the metabolite profile in sheath was mainly characterised by relatively higher proportion of the sugars and formate. No clear effects of leaf age and other experimental conditions (NT, ET, and LW) were found on the metabolite levels.    Relative abundance in a clone library (%) a Atmospheric conditions Temperature a * and ** indicate statistical significance at the 1 and 5% levels (P < 0.01 and P < 0.05), respectively, calculated with the Library Compare of RDP II, between NT sample under ambient atmosphere and other samples. Suc (sucrose), the bucket containing Suc, GLs, and CGA, were colored as shown 7 on the right. 8