Plant Shoots

Amino acids were extracted by homogenizing plant shoots (200 mg FW) in 1 ml of 80% (v/v) aqueous ethanol (MagNALyser; Roche, Vilvoorde, Belgium; 1 min, 7000 rpm), spiked with norvaline to estimate the loss of amino acids during extraction, and centrifugation at 20 000 g for 20 min. The supernatant was vacuum‐evaporated, and the pellet resuspended in 1 ml of chloroform. The plant residue was re‐extracted with 1 ml HPLC grade water using MagNALyser and the supernatant after centrifugation (20 000 g for 20 min) was mixed with the pellet suspended in chloroform. Then the extracts were centrifuged for 10 min at 20 000 g and the aqueous phase was filtered by Millipore microfilters (0.2‐μm pore size) before assaying amino acid concentrations. Amino acids were measured by using a Waters Acquity UPLC‐tqd system (Milford, MA, USA) equipped with a BEH amide 2.1 × 50 column (Sinha et al., 2013).

The entire shoots of three week old Arabidopsis plants (Col-0 ecotype), grown under continuous light, were harvested, frozen in liquid nitrogen and ground to powder (1 g). Plant tissue was resuspended in 10 ml of HBM buffer (25 mM Tris-Cl pH 7.6, 440 mM sucrose, 10 mM MgCl₂ and 0.1% Triton-X, 10 mM β-mercaptoethanol, 2 mM spermine, 1 mM PMSF, 1 ug/ml pepstatin and EDTA-free protease inhibitor cocktail (Roche), homogenized and filtered through Miracloth (Calbiochem). After spinning at 3000 rpm for 5 min (SS-34, Sorvall), the pellet was resuspended in 5 ml of NIB buffer (20 mM Tris-Cl pH 7.6, 250 mM sucrose, 5 mM MgCl₂, 5 mM KCl, 0.1% Triton-X, 10 mM β-mercaptoethanol), applied to a 15/50% Percoll (GE Healthcare) gradient in NIB and spun 2000 rpm for 20 min (SS-34, Sorvall). Isolated nuclei were washed two times in NIB buffer and flash frozen in liquid nitrogen in HBC buffer (25 mM Tris-Cl pH 7.6, 25 mM Tris-Cl pH 7.6, 440 mM sucrose, 10 mM MgCl₂ and 0.1% Triton-X, 10 mM β-mercaptoethanol, 20% glycerol). Nuclei from 1/5 of each preparation were treated with four ul of RNAse A, 10 ug/ul, (Qiagen) and used for Micrococcal Nuclease (Takara) digestion for 6 minutes (final concentration 0.2 U/ul) in digestion buffer (16 mM Tris-Cl, pH 7.6, 50 mM NaCl, 2.5 mM CaCl₂, 0.01 mM PMSF and EDTA-free protease inhibitor cocktail (Roche) and stopped with 10 mM EDTA. Mononucleosomes were released by treating nuclei with 0.1% Triton-X for 1–2 hours in the cold, and then pelleting the debris by centrifuging at 3500 rpm for 3 min (Eppendorf, 5415R). 500 ul of supernatant was applied to 50 ul of Dynabeads Protein A (Invitrogen) that were preincubated with 2.5 ug of the appropriate antibody (#1220, monoclonal anti-H3K9m2 antibody, Abcam; #1791, polyclonal anti-H3 antibody, Abcam) in buffer (20 mM Tris-Cl, pH 7.6, 50 mM NaCl, 5 mM EDTA and 0.1% Triton) and incubated overnight. Beads were washed (10 min incubation in the cold) with 500 ul of the following buffers: 50 mM Tris-Cl pH 7.6, 10 mM EDTA, 0.1 mM PMSF, protease inhibitor cocktail (Roche) with changing concentration of NaCl to 50 mM, 100 mM and 150 mM, subsequently. Final wash was done in TE buffer, without incubation and immunocomplexes were eluted with 500 ul of 0.1% SDS and 0.1 M NaHCO₃ at 65°C for 10 min. DNA was then purified using conventional phenol-chlorophorm extraction and ethanol/salt precipitation. DNA amplification was performed using the GenomePlex® Whole Genome Amplification Kit (Sigma). Four amplification reactions were performed in parallel for each sample to minimize spurious amplification artifacts and purified using QlAquick spin columns (Qiagen) and the products were combined. 4–6 ug of DNA was obtained and Roche Nimblegen performed array hybridization, washing and scanning. For the ChIP with crosslinked tissue, a previously described protocol was used [45] (link).

To isolate cultivable bacteria, surface sterilized root and shoot tissue of three plants were separated and transferred to mortars with 5 ml sterile 10 mM MgSO₄ to compose a mixed sample. The plant tissues were separately crushed and serial dilutions were prepared (0, 10⁻¹, 10⁻², 10⁻³, 10⁻⁴). One hundred microlitre of each dilution was applied to the Petri dishes containing different media (Table 1); all Petri dishes were established in triplicate. The Petri dishes were incubated at 30°C for 4 days after which the cfu per gram of fresh plant tissue was determined; the averages and standard errors were calculated for each treatment. The colonies were then purified and 584 strains were stored at −80°C in glycerol (15%_w glycerol, 0.85%_w NaCl).

Soybean saplings' plant shoots and roots as well as the 30-day-old fresh rosette leaves of A. thaliana were chosen to measure HMGR enzyme activity. The 0.1 g samples were collected and used for enzyme assays according to the protocol described by the Plant HMG-CoA reductase (HMGR) ELISA Kit (Wuhan Chundu Biotechnology, China).

Plant and soil samples were taken at the elongation, heading, and ripening. Since climatic change treatment altered wheat development, soil and plant sampling was conducted based on the phenological stage. Wheat plants were randomly collected 1 m² from each plot, plant sample was separated into shoot and root. Root samples were rinsed with water to get rid of the soil. Meanwhile, the shoot and root samples were rinsed and de-enzyme at 105°C for 0.5 h, and then oven-dried at 70°C for 48 h. All plant samples were ground to 0.25 mm. For soil samples, five soil cores (0-15 cm) were taken and then homogenized to form a mixed soil sample. After removing visible residues and stones, soil samples were passed through a 2 mm sieve and maintained at 4°C before analysis.
Soil available P was extracted with NaHCO₃ and analyzed by a spectrophotometer (TU-1810, China). Soil available K concentrations were estimated using a flame photometer (FP6410, China) after extraction with 1 M ammonium acetate (NH₄OAc). Soil ${\text{NH}}_4^{+}$ -N and ${\text{NO}}_3^{-}$ N concentrations were determined using a subsection flow analysis instrument (Skalar, Netherlands).
Plant shoot and root samples were pretreated with H₂SO₄-H₂O₂, and analyzed for N concentrations by the Kjeldahl digestion method. Phosphorus concentrations were determined by a spectrophotometer (TU-1810, China), and K concentrations by a flame photometer (FP6410, China).

We measured precipitation, temperature, and canopy openness as environmental conditions that might affect the dependency of C. goeringii against mycorrhizas. Precipitation and temperature were measured as the macro-environmental factors which might affect all individuals in the site equally, whereas canopy openness was measured as the micro-environmental factors which might affect each individual plant. The precipitation and temperature were measured by the Meteorological equipment of the Ecological Garden. The total precipitation and the average temperature over 10 d before the sampling date, respectively, were utilized (both values included the values on the sampling date). The photos of the canopy above each plant individual were taken by a built-in camera of a smartphone (AQUOS sense lite SH-M05; SHARP, Japan) equipped with a fisheye lens (Daiso, Japan), which were imported to CanopOn 2 (http://takenaka-akio.org/etc/canopon2/) to major canopy openness. To index the maturity of each individual of C. goeringii, the number of pseudobulbs was recorded. Because C. goeringii has a sympodial growth and makes new shoots (pseudobulbs) once a year, the number of the pseudobulbs reflects the plant age after shoot formation.