Barley growth & exudate collection
1) Surface sterilise barley seeds washing them in 70 % ethanol for 30 s and then in 5 % sodium hypochlorite for 15 min; Rinse thoroughly with sterile ddH2O.
2) Place surface sterilised seeds on petri dishes containing a semi-solid 0.5 % agar solution maintained at room temperature to promote germination (aprox. 2 days).
3) Transfer barley seedlings displaying a comparable development into a 400 mL plastic pot filled with 300 g of pre-sterilised silver sand. We recommend 3 seedlings per pot and an equivalent number of pots per genotype and unplanted controls.
4) Arrange the pots in a randomised blocks design (e.g., n=15 pots per genotype/block) in a glasshouse at 18/14 °C (day/night) temperature regime with 16 h daylight supplemented with artificial lighting to maintain a minimum light intensity of 200 µmol quanta m−2 s−1.
5) Water both planted and unplanted pots with 50 mL of a modified Hoagland’s solution9 designated N100, and containing all the macro and micro-nutrient required for optimum barley growth10 at intervals of 48 hours.
6) In the last week of growth (i.e., the second week or the third week, respectively) replace the N100 solution another one designated N25, containing a quarter of the total nitrogen compared to the former one10.
7) After 2 and 3 weeks (harvesting timepoints) and following the randomised block design, gently uproot the plants from the pots and wash off the sand around the roots using water with a final wash in sterile ddH2O.
8) Using a plastic jar (at least 100 mL vol), submerge the washed plant roots in 50 mL of sterile ddH2O and leave them for 6 to 7 h to exudate. Use three plants (i.e., an individual pot) per genotype per jar.
9) Add 50 mL sterile ddH2O to unplanted pots and process the flow-through as planted samples.
10) Upon incubation, collect the root exudates (100 mL) and unplanted controls in clean plastic jars and filter them using fluted filter paper (cellulose Whatman No. 42).
11) Add 100 µL of 2 mg/mL erythritol solution (extraction standard for the GC/MS) to each filtered solution. The erythritol is used as an internal control for the polar fraction and to normalise the GC/MS data, since it is added in a known concentration.
12) Flash-freeze the filtered solutions at -80 °C.
13) The roots are then oven dried for 48 h at 70 °C. The dried root weight is an alternative method to normalise the GC/MS data.
14) Freeze dry the obtained material for 4 consecutive days.
15) At the completion of point 14, > 25 mg per genotype/condition of freeze-dried exudates should be obtained.
Sample preparation for elemental analysis and GC/MS
1) Process an aliquot of freeze-dried exudates and unplanted wash-through (5 mg) with the Elemental analyser for total carbon and nitrogen quantification using the Dumas method11.
2) Important: prepare risk assessments for each of the hazardous substances to be used in this part of the protocol.
Day 1: experimental preparation
3) Use an aliquot of 20 mg of the same samples to perform a semiquantitative gas chromatography–mass spectrometry (GC/MS) analysis according to reference12 and described below:
4) Glassware preparation: all glassware must be washed consecutively with three times tap water, one time with 100% methanol and one-time with 100% chloroform (Figure 2) before use. For example: preparation for 24 samples + 2 blanks: 26 culture tubes + lids; 80 Quick fit test tubes + PTFE-lined caps; 60 amber glass vials (8 mL) + PTFE-lined caps; 4 volumetric flasks + lids; 4 beakers (Figure 2)
5) Prepare the internal standard solutions (polar and non-polar) and the retention standard solutions. These standard solutions should be kept in the fridge until used. Sonicate these solutions before use.
6) Weight 20 mg (+-0.5 mg) of the samples into a tared culture tube (150 x 16 mm), avoiding electrostatic forces as freeze-dried samples have low density. Record the exact sample weight (e.g. 20.34 mg) as this exact weight will be used for downstream GC/MS data normalisation.
Day 2: separation of polar and non-polar fractions
7) Sonicate the internal standards and switch on the 30 oC incubator.
8) Add methanol (3 mL) to the each of culture tubes containing your weighed sample and incubated at 30 oC for 30 minutes at 1500 rpm . Make sure that culture tubes lids remain in place while shaking.
9) Add 100 µl of each polar (ribitol) and non-polar (n-nonadecanoic acid methyl ester) internal standard solutions to samples.
10) Add deionised water (0.75 mL) and shake at 1500 rpm at 30 oC for 30 min.
11) Add chloroform (6 mL) and shake at 2500 rpm at 30 oC for 30 min.
12) Add water (1.5 mL) and shake vigorously by hand for several seconds.
13) Centrifuge tubes on centrifugal evaporator (1200 rpm, 10 min, no heating, no vacuum).
14) Separate the polar (upper layer) and non-polar (lower layer) fractions into separate amber glass vials (8 mL) using a Pasteur pipette. This step needs to be carefully performed as it is important to do not have cross-contamination of the fractions (Figure 3).
15) Store the polar and non-polar extracts in a -20 oC freezer until they are ready for derivatisation.
Day 3: polar and non-polar fractions derivatisation
16) Sonicate the retention standard solutions for 10 minutes and switch on the 50 oC and 30 oC incubators.
17) Remove polar extract from freezer, allow it to warm up to room temperature.
18) Pipette polar fractions (250 µL) into culture tubes Quick fit (100 x 16 mm) from the upper part to avoid residual non-polar fraction.
19) Transfer the tubes to a centrifugal evaporator (ensure that caps are not mixed in between the samples) and evaporate the solvent (about 45 minutes, ensure they are dry) (Figure 4)
20) In the meantime, prepare a solution of methoxylamine hydrochloride (20 mg; 98 %) in anhydrous pyridine (1 mL). Pyridine is very hygroscopic, Parafilm seal and keep it in a desiccator. At this stage we may need to sonicate the solution to make sure that the methoxilamine is well dissolved.
21) Oximation: add the methoxylamine hydrochloride solution (80 µL) to the dried polar fractions and incubate at 50 oC for 4 hours..
22) Add MSTFA (80 µL) to tubes, and incubate for 30 min at 37 oC. The polar samples are now derivatised.
23) Add the retention standard solution (50 µL) to the amber autosampler vials (300 µL fixed glass inserts with PTFE coated snap caps) and allow the isohexane to evaporate in the vials at room temperature (use a nitrogen pump carefully to accelerate the process). Prepare at least 5 additional vials just in case they break.
24) Add the derivatised polar fractions (40 µL) to amber autosampler vials then add dry pyridine (40 µL). The polar samples are ready now for GC/MS.
25) Add the non-polar fractions (4 mL) into culture tubes Quick-fit (100 x 16 mm). Pipette from the lower part to avoid any residual polar fraction.
26) Dry the non-polar fraction in the centrifugal evaporator (30 min or longer until dry, pulsing but no heating), if required.
27) Transesterification: Add chloroform (1 mL) and methanolic sulphuric acid (2 mL). Methanolic sulphuric acid (1 mL concentrated sulphuric acid in 99 mL methanol, freshly made up). First add the methanol and then sulphuric acid CAUTION: dilute slowly the concentrated sulphuric acid into the methanol.
28) Incubate overnight (>16 h) at 50 oC.
Day 4: non-polar fractions derivatisation (continuation)
29) Sonicate the retention standard solution and switch on the 37 oC incubator
30) Remove tubes with the non-polar fractions from the incubator and bring them to ambient temperature
31) Add aqueous sodium chloride (5 mL; 5 % (w/v)) and chloroform (3 mL) to each tube, shake vigorously and allow layers to settle.
32) Remove the top aqueous layer and discard.
33) Add aqueous potassium hydrogen carbonate (3 mL; 2 % (w/v) to the lower chloroform:methanol layer, shake vigorously and allow layers to settle.
34) Remove the top aqueous layer and discard.
35) Pipette the lower chloroform:methanol phase through freshly prepared columns of anhydrous sodium sulphate in granules. These columns are prepared in cotton wool plugged Pasteur pipettes and pre-washed with chloroform (4 mL)). This step avoids aqueous phase remains in the non-polar fraction (Figure 5).
36) Pass additional chloroform (2 mL) through the column and collect with fractions.
37) Dry the fractions down in the centrifugal evaporator (60 min or until dry, no heat with pulsing).
38) Add chloroform (50 µL), anhydrous pyridine (10 µl) and MSTFA (40 µL) and heat at 37 oC for 30 min in an incubator. The non-polar samples are now derivatised.
39) Add the retention the standard solution (50 µL) to the amber autosampler vials (300 µL fixed glass inserts with PTFE coated snap caps) and allow the isohexane to evaporate in the vials at room temperature (use a nitrogen pump carefully to accelerate the process). Prepare at least 5 additional vials in case they break.
40) Add the derivatised non-polar fraction (40 µL) to the vials plus anhydrous pyridine (40 µL). The non-polar samples are now ready for analysis by GC-MS.
1) Acquire metabolite profiles using a GC–MS (DSQII Thermo-Finnigan, UK) system carried on a DB5-MS column (15 m x 0.25 mm x 0.25 µm; J&W, Folsom, CA, USA). Inject samples (1 µL) into a programmable temperature vaporising (PTV) injector with a split of 80:1.
2) Use the following PTV conditions: injection temperature 132 °C for 1 min, transfer rate 14.5 °C.s-1, transfer temperature 320 °C for 1 min, clean rate 14.5 °C.s-1 and clean temperature 400 °C for 2 min.
3) Perform the chromatography using helium at 1.5 mL/min in constant flow mode. Set GC temperatures at 100 °C for 2.1 min, 25 °C/min to 320 °C, then isothermal for 3.5 min. Set the GC–MS interface temperature at 250 °C.
4) Acquire mass spectra under electron impact (EI) ionisation conditions at 70 eV over the mass range 50–900 a.m.u at six scans per second with a source temperature 200 °C and a solvent delay of 1.25 min.
5) Set acquisition rates to give approximately ten data points across a chromatographic peak. Data were acquired using the XCALIBUR (Thermo Scientific, Waltham, MA, USA) software package V. 2.0.7.
6) Perform semi-quantification of data by integrating selected ion chromatographic peaks.