Chapter 2: Materials and Methods

2.1 Plant materials

2.1.1 Growth of crop species prior to hydroponics

Wheat (Triticum aestivum L. cvs Avalon, Cadenza and Skyfall), maize (Zea mays L. F1 cv. Earlibird), barley (Hordeum vulgare L. cvs Golden Primrose and Pallas Andrew), pea (Pisum sativum L. cv. Avola), rapeseed (Brassica napus L. cv. Extrovert) and tomato (Solanum lycopersicum L. cv. Ailsa Craig) were initially grown in a Sanyo growth cabinet (MLR-352-PE; Sanyo, Japan). Seeds were not sterilised before planting. Seeds were grown in a mixture of vermiculite and perlite (50:50) for 7 days with a photoperiod of 16 h, a temperature of 22°C and an average light level of 634 µmol m^-1 s^-1. Plants were watered every two days using 300 mL deionised water (dH₂O). Twenty seeds were assigned to one 10 cm plant pot. Seeds were evenly planted on the surface of the vermiculite and perlite, which was then covered with a 2 cm layer of substrate. Plant pots then were watered and placed into the growth cabinet. After 7 days, seedlings were placed into a glasshouse overnight to acclimatise.

2.1.2 Liverwort growth conditions and agar gel extraction

Liverwort growth and agar extraction was performed by Ms Bev Merry. Liverwort (Marchantia polymorpha L.) samples were obtained locally (GPS coordinates 53.804°N/1-.55517°E) in May 2016 by Dr Katie Field. After collection, liverworts were grown on 0.9% plant agar (Biochemie, Netherlands) with full-strength BG11 medium without sucrose (Rippka et al. 1979) within the Sanyo growth cabinet. Liverworts were grown with a photoperiod of 12 h, a temperature of 22°C and an average light level of 634 µmol m-1 s-1. After collection, liverworts were grown on agar by removing each thallus using tweezers under a compound microscope, and placing them individually onto the agar in a 10 cm² Petri dish. These liverworts were grown for two weeks until sub-culturing. Ten liverwort gemmae (cup-like appendage that form clones) were removed using tweezers, and then grown on a Petri dish for 30 days. Some liverworts were grown on 0.9% agar with dH₂O only for 30 days. One Petri dish containing 10 gemmae formed one biological replicate. After 7 days, some liverworts were imaged with a Leica MZ12 light microscope with a GXCAM-5 camera (Leica Microsystems, UK) using a 4x objective. After 30 days of growth, gemmae were removed from agar. Where the gemmae were grown, agar was finely cut, 5 cm² round location of each gemmae, into small cubes and placed into a 15 mL Falcon tube with 10 mL dH₂O. The agar gels with the water were agitated on a see-saw rocker at 50 osc/min (Stuart Gyro-Rocker: SSL4, UK) overnight. After agitation, liquid was removed and stored in a fresh 15 mL Falcon tube at -20°C until assaying.

2.2 Hydroponic system for the isolation of polysaccharides released by roots

Seedlings grown in vermiculite and perlite (as previously outlined in section 2.1.1) were transferred to the hydroponic system, which used 9 L half-strength Hoagland’s solution (7.1 g of H2395-10L; Sigma-Aldrich, UK; Arnon 1938). Twelve plants were assigned to each 9 L bucket to form one biological replicate. A standard of three biological replicates of each species and cultivars was grown within a hydroponic system (Figure 2.1). The plants were grown for a further 14 days until harvesting. The glasshouse had a 16 h photoperiod with a constant temperature of 22°C, and an average light level of 1,382 µmol m^-1 s^-1. A constant air flow (1 L/min) was added to the medium using an air compressor (12 v; Corning Limited, UK) with a limit of four buckets per compressor. In order to transfer the plants into the hydroponic system, the plants pots were tipped onto their side with the plants gently being removed from the substrate. After removing plants, the roots were gently rubbed down using a thumb and index finger to remove any loose vermiculite and perlite. Two plants were assigned to each foam holder by placing the foam holder around the base of the stems. The foam holder with the plants was fitted into the hole of the polystyrene cover. Every two days each hydroponic bucket was topped up with dH₂O if the medium level had dropped below 9 L.

After 14 days of growth, plants were harvested, and their total fresh root weights and longest root lengths were measured. The 9 L buckets containing the hydroponate (hydroponic medium with plant exudate) were concentrated on the same day of harvest. Post-harvest, all hydroponic equipment including buckets, aerating tubing, polystyrene covers were thoroughly cleaned using household bleach (Domestos, UK), and stored until further use. The foam holders were autoclaved for 45 minutes at 121°C using a benchtop autoclave (Prestige Classic Medical, UK).

Figure 2.1 I Hydroponics system developed for growing crop species

Crops were grown for one week in 50:50 mixture of perlite and vermiculite prior to hydroponic culture. After pre-hydroponic growth, plants were transferred into 9 L ½ strength Hoagland's solution. Twelve plants were grown in each bucket. An air compressor constantly pumped air into the hydroponic medium. Foam holders prevented plants from falling, and prevented the hydroponic medium being exposed to the atmosphere. This hydroponics system was placed within a glasshouse, which had a 16 h photoperiod with a constant temperature of 22°C, and an average light level of 1,382 µmol m-1 s-1. After two weeks of growth, plants are harvested and the hydroponic medium concentrated. Images included are from an open source platform. Numbers on above view indicated where each pair of plants were placed.

2.3 Concentrating hydroponic media

After 14 days of growth, hydroponates were harvested and concentrated using an ultrafiltration system. To determine which filter cut-off point to use to concentrate the released polysaccharides, a range (100 KDa, 50 KDa, 30 KDa and 10 KDa) of Vivaspin 2 mL tubes (Sartorius, Germany) were used. One millilitre of hydroponate was pipetted into a tube, which was subsequently spun down for 5 min at 3,893 x g.

Prior to concentrating, the hydroponates were filtered using pre-folded paper filters (Whatman Grade 2V, 240 mm; GE Healthcare, Germany) to prevent any particles, such as plant debris, from blocking the concentrator. After filtering, the hydroponates from each bucket were not pooled but kept as biological replicates. A Centramate ultrafiltration system (FScentr005K10; PALL Life Sciences, US) which used a 30 KDa cut-off point cassette was utilised to concentrate each 9 L volume to ~200 mL. An Easy-Load pump (Masterflex, US) was used to circulate the liquids within the ultrafiltration system (Figure 2.2). Prior to use, the cassette was stored in 0.1 M NaOH which was cleared using 5 L dH₂O. This was performed by securing the cassette into the ultrafiltration system by tightening the screws (torque 10 Nm), and then placing the inlet tube into the water, and the retentate and filtrate tubes into a large container kept on the floor. The water was run through the cassette using a flow rate of 200 mL/min. After passing the water though the system, the pH was measured by removing the retentate and filtrate tubes individually, and placing the pH paper onto the flow of water. Once the pH had become neutral the ultrafiltration system was ready to use.

The inlet and retentate tubes were placed and secured in the hydroponate with the filtrate tube being retained in the container on the floor (Figure 2.2). The container holding the hydroponate was covered using aluminium foil. A flow rate of 500 mL/min (1 bar) was set with each sample being concentrated from 9 L to 200 mL. After concentrating, the hydroponates were dialysed in dH₂O (200x sample volume) for 3 days, with two changes per day, at RT using a 3.5 KDa cut-off point. Spectra/pro membrane (Spectrumlabs, US). The subsequent dialysed concentrated hydroponates were transferred to 4 x 50 mL Falcon tubes (Falcon, US), which were placed at -80°C for 24 h. After 24 h, concentrated hydroponates were freeze-dried using a lyophiliser (Heto LyoPro 6,000, US) for 4 days at -90°C. The dried concentrated hydroponates were transferred to 15 mL Falcon tubes.

Once the ultrafiltration system had concentrated each 9 L hydroponate the system was washed using 5 L dH₂O with a flow rate of 500 mL/min. The inlet tube was placed into the water with the retentate and filtrate placed into the container kept on the floor. This washing step was repeated between each biological replicate. A further washing step was required between each species and cultivar. For this additional step, 0.5 M NaOH was run though the system for 5 min after the water was passed through. A further 0.1 M NaOH was run through the system for 15 min. Once complete, 5 L dH₂O was passed through the system. When the pH returned to neutral the next hydroponates were concentrated. When the ultrafiltration system was no longer going to be used, the above step was repeated without the final wash of 5 L dH₂O, and was stored in 0.1 M NaOH at 4°C until future use. The cassette was also stored in 0.1 M NaOH at 4°C.

Figure 2.2 I Hydroponate was concentrated by 45x using the ultrafiltration system

Hydroponate was passed through a 30 KDa cut-off point cassette, which was circulated through the inlet and retentate tubes (red). Any molecules that were less than 30 KDa was passed through the filtrate tube (green). Direct hydroponate was passed through the ultrafiltration system at 500 mL/min with 1 bar of pressure. Once 9 L of direct hydroponate was concentrated to 200 mL, the cassette was purged with dH2O and NaOH by removing the retentate tube and placing it with the filtrate tube.

2.4 Enzyme Linked Immuno-Sorbent Assay (ELISA)

ELISA was performed (Engvall and Perlmann 1971) using 96 well microtitre plates (NUNC Maxisorp, Thermo Fisher Scientific, Denmark). Samples were dissolved in 1x phosphate buffered solution (PBS; Severn Biotech Limited, UK) as standard for coating. During titration the bottom row of each microtitre plate did not contain any sample; this was to act as a no antigen control. All microtitre plates were incubated at 4°C overnight.

After overnight incubation, plates were hand washed using tap water by shaking them three times under water. After washing, plates were dried by tapping them on a small pile of dry paper towels. Plates were incubated using 300 µL per well of MP/PBS (5% w/v milk powder (Marvel, UK) in 1x PBS that had been filtered using a fine tea strainer to remove milk aggregates) for 2 h at room temperature (RT). The plates were double wrapped in aluminium foil during each incubation. After incubation, the washing step was repeated. Subsequently, the plates were incubated with a 1:10 dilution of a chosen anti-rat MAb (hybridoma cell supernatant; Table 2.1) in MP/PBS (100 µL per well) for 1 h at RT. The washing step was repeated with the plates being shaken six times under water before drying. After washing, a 1:1,000 dilution of anti-rat secondary antibody coupled with horse radish peroxidase (HRP; A9552; Sigma-Aldrich, US) secondary antibody was added to MP/PBS (100 µL per well) for 1 h at RT. For Complex Carbohydrate Research Centre (CCRC) antibodies, a 1:1,000 dilution of anti-mouse coupled with HRP was used (A9044; Sigma-Aldrich, US). For CBMs, a 1:100 dilution of anti-polyHistidine peroxidase (A7058; Sigma-Aldrich, US) was used followed by an additional incubation step, which used a 1:1,000 dilution of anti-rat coupled with HRP. The washing step was repeated after incubation. The following substrate (100 µL per well) was added to the wells for 8 min: 9 mL dH₂O, 1 mL 1 M sodium acetate pH 6.0, 100 µL 3,3’,5,5’-tretamethylbenzidine (10 mg/mL Dimethyl sulphoxide, DMSO; T-2885; Sigma-Aldrich, US) and 10 µL H₂O₂. After 8 min, 50 µL 2.5 M sulphuric acid was added to the wells. The plates were subsequently read using the absorbance at 450 nm in a Multiskan FC plate reader (Thermo-Scientific, US) that used SkanIt software (Thermo-Scientific, US).

2.5 Sandwich-ELISA analysis

Microtitre plates were coated with 100 µL of 10 µg/mL of the capturing probe CBM2b1-2 (xylan-binding CBM; McCartney et al. 2006) or 10 µg/mL CBM3a (Crystalline cellulose/xyloglucan-binding; Hernandez-Gomez et al. 2015) in 1x PBS. For negative controls, wells just containing 100 µL 1x PBS were coated. Both treatments were doubled wrapped in aluminium foil and incubated overnight at 4°C (Cornuault and Knox 2014). After incubation, plates were washed using tap water by shaking them three times under water. To dry the plates, plates were tapped on a small pile of paper towels. Plates were incubated with 250 µL MP/PBS in each well (Cornuault and Knox 2014). Plates were then doubled wrapped in aluminium foil (this occurred during each incubation), and incubated for 3 h at RT. After incubation washing was repeated. Once plates were dried, MP/PBS with dissolved concentrated hydroponate was pipetted (100 µL per well) into the wells. The plates were then incubated at 4°C overnight. After incubation washing was repeated. Plates were then incubated using a 1:10 in dilution of anti-rat MAb (Table 2.1) in MP/PBS (100 µL per well) for 2 h at RT. After incubation washing was again repeated. Plates were then developed following ELISA as outlined above.

2.6 Tissue printing on nitrocellulose

The nitrocellulose printing and imaging was performed by Ms Sue Marcus. After liverworts were grown (as previously outlined in section 2.1.2) they were removed from the agar using tweezers. A nitrocellulose sheet was then placed on top of the agar, ensuring that the surface area was fully covered. The nitrocellulose and agar were incubated overnight at 4°C uncovered in aluminium foil. After overnight incubation, nitrocellulose was gently washed with tap water. Nitrocellulose was then blocked with MP/PBS with 0.0025% sodium azide to inhibit bacterial growth. The sheet was then incubated at RT for 1 h. After 1 h, a 1:5 dilution of LM25 was added to the nitrocellulose, and incubated for a further 1 h. After incubation, nitrocellulose was gently washed with tap water, and washed again in 1x PBS three times for 5 mins each. Subsequently, block with 1:1,000 dilution of anti-rat HRP was added to the nitrocellulose, and incubated for a further 1 h. Washing was repeated with tap water and 1x PBS. After washing, the following substrate was added to the nitrocellulose, and incubated for 30 min: 25 mL dH₂O, 5 mL chloronaphthol (5 mg/mL in EtOH) and 30 µL of H₂O₂, which was added at the last minute. Once developed, the nitrocellulose was thoroughly washed in a container full of dH₂O for 5 min. Nitrocellulose was then dried in between two sheets of blotting card for 30 min. Subsequently, nitrocellulose was imaged using an Epson Perfection V750 Pro scanner (Epson, Japan).

2.7 Isolation of polysaccharides from root body cell walls

Root cell walls of wheat cultivar, Cadenza were isolated by preparation of Alcohol Insoluble Residue (AIR; Fry, 1988). After weighing fresh root material, the material was immediately placed into liquid nitrogen for 2 min. After liquid nitrogen, the root material was freeze-dried for 3 days prior to AIR. Fifteen milligram of freeze-dried root material was placed into a 2 mL Qiagen tube with two iron beads. The tube was then frozen in liquid nitrogen. The tube was then homogenised into a Tissue lyser (Qiagen, UK) for 2 min at 50 Hz. After grinding, 10 mg of ground root material was placed into a 2 mL Eppendorf tube along with 1 mL 70% EtOH (v/v), which was agitated on a see-saw rocker at 50 osc/min for 1 h. The samples were centrifuged for 1 min at 400 x g. After centrifugation the supernatant was removed. Subsequently, 1 mL solutions of 80%, 90% and 100% EtOH, 100% acetone, and methanol and chloroform (2:3) were added to the samples and each agitated for 1 h at 50 osc/min. Centrifugation was repeated after each incubation. After the methanol and chloroform was added, the samples were left within a laminar flow hood overnight at RT to dry.

One milligram of dried root AIR was placed into a fresh 2 mL tube with 400 µL 4 M KOH and 1% NaBH₄. The tube was then placed into the Tissue lyser for 20 min at 50 Hz. The sample was centrifuged at 3,893 x g for 15 min. Subsequently, the supernatant was removed and placed into a 5 mL Eppendorf tube. The sample was then diluted by 10 times in 1x PBS. The diluted supernatant was neutralised using 80% (v/v) acetic acid. The pH was tested by using pH paper. The neutralised samples were dialysed and freeze-dried as outlined in concentrating hydroponic media. The resulting solutions then were assayed by ELISA, anion-exchange EDC and sandwich-ELISA.

Back to top

2.8 Chromatography analyses of concentrated hydroponates

2.8.1 Anion-exchange Epitope Detection Chromatography (EDC)

EDC analyses were performed by injecting samples, which had been dissolved in 20 mM sodium acetate (pH 4.5) buffer into separate 1 mL weak anion-exchange chromatography columns (Hi–Trap ANX FF, GE Healthcare, UK). A Bio-Rad BioLogic LP System (Bio-Rad, US; Cornuault et al. 2014) was used to undertake the anion-exchange chromatography. Two gradients, step and neutral, of NaCI were used to elute the samples at 1 mL/min.

For gradient 1, Buffer A (20 mM sodium acetate pH 4.5) eluted the samples for 13 min, a linear gradient from 0-20% using Buffer B (50 mM sodium acetate + 0.6 M NaCI) was used for 16 min, 30% Buffer B for 16 min, 40% Buffer B for 16 min, 50% Buffer B for 16 min, followed by 100% Buffer B for 19 min. In order to isolate sufficient fractions for detailed biochemical analysis, gradient 1 was scaled up by fifteen times, using a 15 mL column. This resulted in the programme running Buffer A for 540 min, followed by a linear gradient from 0% to 100% using Buffer B for 540 min, followed by a further 360 min of 100% Buffer B; each fraction became 15 mL.

For gradient 2, samples were eluted with Buffer A for 36 min, followed by a linear gradient from 0% to 100% using Buffer B for 72 min, followed by a further 24 min of 100% Buffer B.

For both gradients, 96 fractions of 1 mL volumes were collected within a 96 well megablock (2.2 mL wells; Sarstedt, Germany). The collected fractions were adjusted to pH 9.0 by adding 40 µL 1 M sodium carbonate into each fraction (1 mL), and thoroughly mixing using an automated pipette. This was confirmed by removing 2 µL of five randomly selected fractions, and pipetting them onto pH paper. Aliquots (100 µL) of each 1 mL fraction were incubated per well on a microtitre plate (Cornuault et al. 2014), and were developed using ELISA. The BioLogic LP data view software (Bio-Rad, US) was used to monitor and log the conductivity of the NaCI gradient during chromatography. Between each run, the column was washed by reducing the salt levels with Buffer A and by injecting 1 mL 0.1 M NaOH. Once NaOH was injected, the column was equilibrated using Buffer A. Once the columns had been washed they were stored at 4°C wrapped in aluminium foil. The tubing was kept in 20% EtOH prior to each use. For the start of each run, the tubing was purged using dH₂O with a flow rate of 6.5 mL/min (Cornuault et al. 2014). After purging, the column was added to the system. Once the column was in place, Buffer B was added at 1 mL/min to increase the salt concentration within the column. After the gradient of salt had reached its maximum peak, Buffer A was added to lower the salt concentration (Cornuault et al. 2014). When the salt concentration was lowered the column had been calibrated for use.

2.8.2 Size-exclusion EDC

A modified version of size exclusion chromatography was followed (Pfannkoch et al. 1980). Samples were each dissolved into 1 mL of vacuum filtered (0.45 µm pore size; Supelco Nylon 66 membrane; Sigma-Aldrich, US) 20 mM sodium acetate with 1 M NaCI, pH 4.5 (buffer). This buffer was injected into a 120 mL HiPrep (16/60 Sephacryl S400H; GE Healthcare, UK) size exclusion chromatography column. An AKTA start chromatography system (GE Healthcare, UK) was used. Unicorn 5.0 Manager Software (Amersham Biosciences Corp, US) was used to monitor column conductivity. Prior to use the column was cleaned by selecting the lowest flow through rate, passing 20 mM sodium acetate with 1 M NaCI pH 4.5 through the column overnight at a flow rate of 0.2 mL/min. After cleaning, 2.5 mL of sodium acetate buffer was passed through the loop where the sample would be injected. Subsequently, samples, dissolved in 1 mL of buffer, were injected into the column with a flow rate of 0.7 mL/min. Ninety-six fractions of 1 mL were collected in total. After size exclusion, the pH of the collected fractions was adjusted to pH 9.0 by adding 40 µL 1 M sodium carbonate. One-hundred microliters of each fraction was added into each well of a 96 microtiter plate. The microtitre plate was incubated overnight at 4°C. A standard ELISA protocol followed, as previously outlined, with the exception of a 1:20 dilution for all MAbs used. After size exclusion, the column was eluted with 20% EtOH at 0.2 mL/min overnight in order to clean it. The column was then wrapped in aluminium foil and stored at 4°C until future use.

A gel filtration HMW and LMW calibration kit (28-4038-42; GE Healthcare, UK) which contained the following protein standards: carbonic anhydrase 29 KDa (bovine erythrocytes), conalbumin 75 KDa (chicken egg white), aldolase 158 KDa (rabbit muscle), ferritin 440 KDa (horse spleen) and thyroglobulin 669 KDa (bovine thyroid) was used to calculate KDa values. Four milligrams of each protein standard was dissolved into 1 mL buffer and individually run on the size exclusion chromatography. Where the signals of protein standards peaked, the KDa values were mapped onto the chromatogram.

2.9 Total carbohydrate content assay

A Phenol-Sulphuric Acid assay was followed to determine the total carbohydrate content of samples (Masuko et al. 2005). Aliquots (50 µL) of each 15 mL fractions from the anion-exchange EDC were removed, and placed into 1.5 mL Eppendorf tubes. Concentrated sulphuric acid (18 M) was rapidly pipetted into the 50 µL sample followed by 30 µL 5% (w/w) phenol in dH₂O. The solutions were then incubated for 5 min at 90°C. The subsequent samples were pipetted into a 96 microtiter plate and read at an absorbance of 450 nm using the plate reader.

2.10 Enzyme digests of concentrated hydroponate

The following enzymes and buffers were made for the enzyme digests: β-xylanase M1 (Trichoderma viride; Megazyme, Ireland) was dissolved in 100 mM sodium acetate (pH 4.5), xyloglucanase (GH5, Paenibacillus sp.; Megazyme, Ireland) was dissolved in 100 mM sodium acetate (pH 5.0) with 0.5 mg/mL BSA. Concentrated hydroponate (1 mg) was dissolved directly into 10 mL of each buffer (as outlined in above). Aliquots (50 µL) containing 50 µg of concentrated hydroponate were further diluted into 950 µL of each buffer. This solution was used for each digest. The β-xylanase M1 digest samples were incubated at 40°C for 2 h. The xyloglucanase digest samples were incubated at 60°C for 2 h. Forty units of xyloglucanase (60 µg of enzyme) was required to breakdown the xyloglucan within the 50 µL sample of concentrated hydroponate. For β-xylanase M1, 480 U (120 µg of enzyme) was required to digest xylan within the concentrated hydroponate. After samples were incubated with the enzymes treatments, the samples were left to cool at RT for 10 min before EDC.

2.11 Monosaccharide composition and monosaccharide linkage analyses

2.11.1 Monosaccharide composition analysis

The monosaccharide composition analysis was undertaken by Mr Bernhard Jaehrig based at the Complex Carbohydrate Research Centre, US. A sample of REC1 (300 µg) was placed into an Eppendorf tube (2 mL), and hydrolysed using 2 M trifluoroacetic acid for 4 h within a sealed tube at 100°C. The trifluoroacetic acid was removed by heat-assisted evaporation, which used a gentle flow of liquid nitrogen. The sample was then reduced in 1 mL (10 mg/mL) sodium borohydride prepared in 1 M ammonium hydroxide, which was incubated for 1 h at RT (Peña et al. 2012b). The resulting residue was removed by adding 300 µL of methanol to the sample. The sample was then dried using heat-assisted evaporation. Aliquots (125 µl) of 9:1 (v/v) methanol in acetic acid were added to the sample for 1 h at RT. Heat-assisted evaporation was repeated (Peña et al. 2012b). The resulting alditol acetates were analysed on a 7890A gas chromatography (Agilent Technologies, US) with a SP2330 bonded phase fused silica capillary column (30 m x 0.25 cm; Sigma-Alchrid, US). The gas chromatography was interfaced to a 5975C Mass Selective Detector (MSD) mass spectrometer that was operating on the electron impact ionization mode (Agilent Technologies, US; York et al. 1985). Inositol (20 µg) was used as an internal standard.

2.11.2 Monosaccharide linkage analysis

The monosaccharide linkage analysis was undertaken by Mr Bernhard Jaehrig based at the Complex Carbohydrate Research Centre, US. A sample of REC1 (1 mg) was suspended in 200 μL of DMSO and agitated (120 rpm) at RT for 1 week. Methylation was undertaken by agitating (120 rpm) the dissolved REC1 in 200 µL of 1 M sodium hydroxide for 15 min, and then in 100 μL of methyl iodide for 45 min, both at RT. An aliquot (2 mL) of dH₂O was then added to the sample. Removal of excess methyl iodide was carried out by sparging small aliquots of liquid nitrogen into the sample (Heiss et al. 2009). Subsequently, the methylated sample was hydrolysed using 400 µL of 2 M trifluoroacetic acid for 2 h within a sealed Eppendorf tube at 120°C. The trifluoroacetic acid was removed by heat-assisted evaporation, which used a gentle flow of liquid nitrogen. Isopropanol (400 µL) was added to the sample and immediately dried using liquid nitrogen. This was repeated twice. The sample was then reduced with the addition of 1.2 mL of sodium borodeuteride prepared in 1 M ammonium hydroxide (10 mg/mL), which was incubated for 3 h at RT. Glacial acetic acid (300 µL) was added to the sample and vortexed. Methanol (300 µL) was then added to the sample, vortexed and dried using liquid nitrogen. This was repeated twice. Three-hundred microliters of 9:1 (v/v) methanol in acetic acid was added to the sample, and vortexed. Heat-assisted evaporation was repeated. Methanol (600 µL) was added to the sample, vortexed and dried. This was repeated twice. Subsequently, the dried sample was acetylated using 250 µL of 16:234 (v/v) dH₂O in acetic anhydride, which was thoroughly agitated to re-suspend the sample (Heiss et al. 2009).

Concentrated trifluoroacetic acid (13 M; 230 µL) was added to the sample, thoroughly agitated, and incubated for 10 min at 50°C. Once cool, 2 mL of isopropyl alcohol was added to the sample, and dried. Two millilitres of 0.2 M sodium carbonate was added to the sample along with 1 mL of dichloromethane, which was vortexed for 30 sec. Centrifugation followed with the top layer of the reaction removed. The bottom layer was washed with dH₂O, centrifuged and removed. This was repeated twice. Heat-assisted drying was repeated. The resulting pellet was dissolved in 1 mL of dichloromethane. The resulting partially methylated alditol acetates were analysed on a 7890A gas chromatography (Agilent Technologies, US) with a SP2330 bonded phase fused silica capillary column (30 m x 0.25 cm; Sigma-Aldrich, US), which separated the polysaccharide products by size. The gas chromatography was interfaced to a 5975C MSD mass spectrometer that was operating on the electron impact ionization mode (Agilent Technologies, US). This mode separated the polysaccharide products by their mass-to-charge (m/z) ratio. Both the retention times determined by gas chromatography and the m/z ratios determined by the mass selective detector were used to calculate the linkages through the Agilent 7890 Series Software (Agilent Technologies, US; Heiss et al. 2009).

2.12 Soil sourcing and preparation

Sandy loam soil for the aggregate analyses was obtained locally (GPS coordinates 53°48'17.4"N 1°33'18.2"W) in May 2015. The soil was collected at a depth of 10 cm. Once removed, all visible containments including plant material and stones were discarded from the test soil. Soil was sieved using a 2,000 µm analytical sieve and sterilised by autoclaving at 121°C for 45 min. For optimal sterilisation, soil was thinly spread on a metal tray. After sterilisation, soil was stored in a large glass beaker, which was doubled wrapped in aluminium foil, and kept the dark at 4°C. To determine soil texture, a typical manual soil texture analysis, widely used in horticulture was undertaken (Brown 2008). Other soil types were sourced and prepared using the same method: clay loam soil (coordinates 53°48'15.3"N 1°33'16.4"W), and slit loam (coordinates 53°48'17.3"N 1°33'18.8"W), both collected in June 2015. Sand (22.5 Kg Blooma Play Sand; B&Q, UK) was also collected and prepared in the same manner. Glacial rock (Fox Glacier, New Zealand; coordinates 43°28'14.4"S 169°56'53.3"E) was collected by Dr Katie Field, and prepared in the same manner.

2.13 Measuring polysaccharide adherence to soils

Commercial polysaccharide standards, gum Arabic from Acacia tree (G975; Sigma-Aldrich, US), xylan from birchwood (P-XYLNBE; Megazyme International, Ireland) and tamarind seed xyloglucan (P-XYGLN; Megazyme International, Ireland), were used. Each standard polysaccharide was added to 1 mg of soil (ratio: 1:100), resulting in a 10 µg/mL solution. After each standard polysaccharide was added, the solution containing both the soil and polysaccharide was agitated for 2 h on a see-saw rocker at 50 osc/min. For a negative control, 10 µg/mL of each commercial standard was placed into tube without any soil. After agitation, samples were centrifuged at 3,856 x g (4°C) for 10 min. Once spun down, supernatant was gently removed and assayed by ELISA. The remaining soil was dried for 48 h at 60°C for dry dispersion analyses, SEM, and immuno-staining. A standard of six technical replicates per analysis was used. This resulted in six rows of wells per plate being used for each group. Results of the replicates were averaged.

To calculate the amounts (µg) of polysaccharide remaining within the supernatant, standard curves were generated (EnCor Biotechnology Inc., 2016) by using commercial polysaccharides (see above) that were titrated (10-fold). Once developed by ELISA, the absorbances were plotted onto a scatter graph (with a maximum of 1.0 OD). The linear part of the scatter graph was removed and re-plotted. Once plotted, the Y and X values of the linear plot were gathered along with R² (minimum value of 0.98 was used). After collecting the outlined values the following formula was used to convert ELISA absorbance into total µg (a), a =((au*Y)-X)*d, where d = dilution factor of the absorbance value used.

2.14 Wet sieving soil analyses of aggregate status

Aliquots (100 mL) of gum Arabic, xylan from birchwood and tamarind seed xyloglucan solutions (10 mg/mL) were added to 100 g of sandy loam soil (ratio: 1:100). These samples were then thoroughly agitated for 2 h at 50 osc/min prior to wet sieving. An Octagon (200 series; Endecotts, UK) mechanical sieve shaker (located in the School of Geography, University of Leeds) was set to 1.8 mm/g for 5 min for each round with a contestant flow of cold tap water. Five sieves were stacked in order of deceasing sizes: 1,000 µm, 500 µm, 250 µm, 90 µm and ≤90 µm. After sieving, sieves were initially dried at 95°C for 30 min. Soil was then transferred using a fine brush into blotting card envelopes that had been weighed before soil was added, and placed at 40°C overnight. After the sieves were cleared of soil they were ready to be used again. Once dried, envelopes with soil were re-weighed, and the weight gained calculated, weight of envelope before soil minus weight after soil. Each fraction was then expressed as a percentage relative to each other. Each treatment was repeated three times; the results were then averaged.

2.15 Dry dispersion analysis of soil aggregation

Dry dispersion particle analyses were carried out using a Morphologi G3 (Malvern, UK) automated particle characterisation microscope. This microscope was based within the Institute of Particle Science and Engineering, School of Chemical Engineering (University of Leeds). Solutions with or without the added polysaccharides (ratio 0.1:100) were agitated for 2 h on a see-saw rocker at 50 osc/min. After agitation, samples were centrifuged (4°C) for 10 min at 3,856 x g. The supernatants were removed and assayed. The pelleted soils were dried for 48 h at 40°C. Soil samples (18 mm³) were added into the dispersal unit within the blast chamber of the microscope. Soil was then dispensed onto a glass slide with a burst (1 bar) of liquid nitrogen. Before and after each analysis the dispersal unit, blast chamber and glass slide were cleaned with antistatic spray. The Morphologi G3 imaged 150,000 particles per soil using a 5x objective. After the microscope had measured all aggregate volumes, any outlining aggregates which did not conform to known aggregate dimensions were manually removed, for instance long strands of fibres or dust particles. The volumes of each aggregate were calculated using the 18.2 Morphologi G3S (Malvern, UK) software, which took three Z-stacks of each aggregate and determined volume (v) by using the following formula, v = 4/3π(d/2)³, where d = diameter of aggregate (µm).

2.16 Scanning electron microscopy (SEM) of sandy loam soil

SEM was undertaken using a Quanta Scanning Electron Microscope (200 FEG; FEI Company, US), which used xT microscope control software (FEI Company, US). For sample preparation refer to dry dispersion analysis of soil aggregation. After soil was dried for 48 h, a thin layer of soil was spread onto a glass slide. Stubs with carbon-rich tape were dipped into the soil. The stubs were immediately coated with a 5 nm thick layer of platinum under a vacuum using a mini sputter coater (SC7620; Polaron Equipment Limited, UK) prior to imaging. Each treatment was repeated three times; representative images were displayed. All samples were imaged with a vacuum pressure of 30 Pa, and with an electrical potential of 3 kV; images of REC1 used an electrical potential of 20 kV.

2.17 Immuno-labelling of soils

Aliquots (100 µL) of dH₂O containing 50 µg of xylan from birchwood and tamarind seed xyloglucan were added to 50 mg of sterile sandy loam (ratio 0.1:100). As controls, soil that had no commercial polysaccharide, only containing dH₂O, was screened with MAbs. Furthermore, soil with the commercial polysaccharide was screened without MAbs. These solutions were agitated for 2 h on a see-saw rocker at 50 osc/min. After agitation, samples were centrifuged (4°C) for 10 min at 3,856 x g. The supernatants were removed. A small scoop (~500 µg) of this soil was then placed into the wells of a Vectabonded eight well microscope slide (MP Biomedicals LLC, Germany), and then gently pressed down. Slides were then covered with aluminium foil, and left to dry overnight at RT. Once dried, excess soil was removed by gently tapping the slide onto a bench.

Wells containing soil were blocked with 10 µL filtered 5% milk powder with 1x PBS for 30 min at RT. Slides were covered during each incubation. A 1:5 dilution of MAb (hybridoma cell supernatant) was directly added to each well, which already contained the block. The slides were then incubated for 1.5 h at RT. MAb solutions were removed, and the wells were each washed three times with 1x PBS for 5 min. After washing, anti-rat (Immunoglobulin G; IgG) coupled with fluorescein isothiocyanate (FITC; F1763; Sigma-Aldrich, US) was added to block using a dilution of 1:100. Aliquots (10 µL) of this solution were added to each well. The slides were incubated for 1 h at RT. The wells were each washed three times with 1x PBS for 5 min. After washing, each well had been treated with 10 µl 0.1% Toluidine Blue O (Sigma-Aldrich, US), which was in 0.2 M sodium phosphate (pH 5.5) for 5 min. Washing was repeated extensively. After washing, a small drop of Citifluor in glycerol/PBS (AF-1; Agar Scientific, UK) anti-fade was placed onto the top of each well before glass cover slides were added. The slides were then imaged using an Olympus Optical GX Microscope (BX61; Olympus, US), Olympus BX-UCB control unit, and a Hamamatsu ORCA publisher camera that were tethered to a PC. An X-Cite 120Q (120 watt; Excelitas Technologies, US) was used as the excitation light source (456 nm). Volocity 4 (PerkinElmer, US) image analysis software was used to merge the resulting bright-field and FITC channels. Each treatment was repeated six times.

2.18 Statistical and phylogenetic tree analyses

The statistical significance of differences between mean or median values was determined using Student’s t-test or the non-parametric equivalent, Mann-Whitney U test. For data sets with three or more groups a One-way Independent ANOVA or the non-parametric equivalent, Kruskal-Wallis test followed by post-hoc Mann-Whitney U tests, were used. Differences were considered significant when the P-values were below 0.05. Minitab 17 (Minitab, UK) statistical software was used for all statistical analyses. For phylogenetic trees, plant genus and species were written in a list format, and pasted onto the PhyloT taxonomy-based phylogenetic tree generator (2015.1; BioByte Solutions, Germany).

Back to top