Influence of cell wall polysaccharides and REC1 on soil properties

5.1 Introduction

One of the aims of this study was to explore possible functions of released polysaccharides in relation to soil aggregate status. For this to occur, commercially equivalent components of the three major polysaccharides detected, AGP, xylan and xyloglucan, as well as the multi-polysaccharide complex, REC1 (Chapter 4), were added to soil. The soil was then assessed using various aggregate stability assays. This investigation uncovered that the commercial polysaccharide standards and subdomains of REC1 could readily adhere to soil, and increased the abundance of macroaggregates when supplemented to the soil. REC1 was the most effective soil aggregator when compared to the polysaccharide standards.

Plants secrete root mucilage to lubricate the root caps and tips to aid penetration through the soil. Once released, the polysaccharides within root mucilage contribute to carbon levels within the rhizosphere. This continual addition of carbon is thought to modify the structure of the surrounding soil (Tisdall and Oades 1982). In order to assess the structure of soil, aggregate status is measured and considered a good indicator of soil health (Kibblewhite et al. 2008). In uncultivated land, aggregates in soil are abundant, being formed of a mixture of organic and mineral particles, which increase the contact that the roots have with the soil and lowers the risk or erosion (Griffiths and Burns 1972; Tisdall and Oades 1982). An increase in the abundance of soil aggregates and strength of the adhesion of particles to the root surface, results in roots being able to extract more water and minerals from the rhizosphere. Other benefits include an increase in aeration, water infiltration and enhanced contact with beneficial soil-dwelling microorganisms, including mycorrhizal fungi (Haynes and Francis 1993; Lal 1997; Kibblewhite et al. 2008). In cultivated land, these aggregates are routinely broken down by systematic ploughing (Six and Paustian 1999; Ji et al. 2013). This agricultural treatment is resulting in ever increasing amounts of soil being eroded, and once productive land being lost (Hamilton et al. 2015).

To assess soil quality and determine how vulnerable a soil is to erosion, several techniques are employed. Various methods of sieving have been developed to examine aggregate stability (Day 1965; De Booth et al. 1984; Cheshire 1990; Brown 2008). One particular sieving method, wet sieving subjects soil to a constant flow of water to assess the water stability of aggregates (Brown 2008). Other techniques include visualising aggregates through optical microscopy (De Booth et al. 1984; Cheshire 1990; Hamilton et al. 2015). A novel application was developed for this investigation, which used an automated particle characterisation microscope. As a part of this dry particle dispersion assay, soil was subjected to high mechanical forces through a burst of liquid nitrogen. This burst projects soil onto a glass slide where an automated particle characterisation microscope calculates the volume of each aggregate. If soil aggregates are strongly associated they will remain bound together after hitting the slide. If weakly associated with each other they will readily break apart after hitting the slide.

5.2 Results

5.2.1 Commercial polysaccharides can adhere to soil

To explore roles of polysaccharides released from plant roots on soil properties, a range of the most commonly available soil types were selected, including sandy, silt and clay loams. Commercially equivalent forms of known polysaccharides released by roots were added to soil to see if they could adhere to a heterogeneous mixture of particles within each soil type. The commercial polysaccharides, gum Arabic (AGP standard), xylan from birchwood (hereon referred to as xylan) and tamarind seed xyloglucan (hereon referred to as xyloglucan), were added (10 µg in 1 mL) to 1 mg sterile soil. The soil with the test polysaccharides was agitated for 2 h. After agitation, soil solutions were centrifuged (3,856 x g) for 10 min. After centrifugation, the supernatant from each sample was removed and screened using ELISA with the following MAbs (1:10 dilution), LM2 (AGP), LM11 (xylan) and LM25 (xyloglucan). Calibration curves were generated to convert the ELISA absorbances from the antibodies to µg/mL.

As expected the negative control (na) where test polysaccharides were only dissolved into dH₂O had the highest amounts detected. This also confirmed that the test polysaccharides were not adhering to the inner surface of the Falcon tubes (Figure 5.1, na). Adherence of the test polysaccharides was variable across the different soil types (Figure 5.1). Xyloglucan could readily adhere to all the soils. On average there was a reduction of 99% of xyloglucan when added to the substrates (One-Way Independent ANOVA, F= 351.12, P= 0.0001). There was a 43% reduction in gum Arabic when added to clay loam soil (One-Way Independent ANOVA, F= 40.32, P= <0.001). For xylan, there was a reduction of 97% when added to sandy loam soil (One-Way Independent ANOVA, F= 252.88, P= 0.0001). Overall, clay loam released the lowest amounts of test polysaccharides when the supernatant was assayed (Figure 5.1). Sand released the highest amounts of test polysaccharides when the supernatant was assayed.

Figure 5.1 I Commercial polysaccharides could adhere to a range of soil types

Xyloglucan was the most effective commercial polysaccharide to adhere to the substrates. The most effective substrate to take up xyloglucan was the sandy loam soil. Gum Arabic was the least effective commercial polysaccharide to adhere to the substrates. However, the most effective substrate to take up gum Arabic was the clay loam soil. Xylan could readily adhere to clay, sandy and silt loam soils. The most effective substrate to take up xylan was the sandy loam soil. Commercial polysaccharides (10 µg in mL) were added to 1 mg of sterilised substrate, and incubated for 2 h. A 1:25 dilution of antigen was used; na = no substrate controls. Data are a mean of three biological replicates. Significant differences are indicated as follows: **P= 0.001 and ***P= 0.0001; standard deviation bars are displayed.

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5.2.2 Xylan and xyloglucan increased the abundance of aggregates

To examine the effects of commercial polysaccharides (xylan and xyloglucan), equivalent to those released by plant roots on soil aggregate status, soil was subjected to wet sieving and dry particle dispersion analysis. One-hundred grams of sterile sandy loam soil was wetted with test polysaccharide solutions (10 mg/mL), and agitated for 2 h. After agitation, soil was added to a series of descending sized sieves, the highest sieve (1,000 µm) was placed on top, and the smallest (90 µm) placed at the bottom. Soil was subjected to a constant flow of tap water whilst being shaken for 5 min. After wet sieving, soil retained in each sieve was dried and weighed. The wet sieving analysis revealed that xylan and xyloglucan increased the abundance of macroaggregates ≥500 µm (Figure 5.2, A; Mann-Whitney U, W= 6.0, P= 0.01). As a result of the larger proportion of macroaggregates, the proportion of microaggregates was significantly lower when xylan and xyloglucan was added to the soil (Mann-Whitney U, W= 6.0, P= 0.01). When gum Arabic was added to the soil there was no significant alteration within the abundance of aggregates compared to the no soil control (Figure 5.2, A).

For dry dispersion analysis, test polysaccharide solutions (1 µg/mL) were added to sterile sandy loam soil, and agitated for 2 h. After agitation, soil was centrifuged with the supernatant removed. Soil was then dried for 48 h. A small scoop (18 mm³) was added to a blast disk, which was placed within a sealed blast chamber. Soil was then projected onto a glass slide with high mechanical force through a burst of liquid nitrogen. An automated particle characterisation microscope determined the volumes of each aggregate that was on the slide. Dry dispersion analysis demonstrated an increase in the abundance of macroaggregates ≥500 µm³ when test polysaccharides were added (Figure 5.2, B, C and D; Mann-Whitney U, W= 595, P= 0.01). This was most apparent when xyloglucan was added, which contained the most macroaggregates (Figure 5.2, D; One-way ANOVA, F= 670.52, P= 0.01). During dry particle dispersion analysis, gum Arabic contained the least amount of macroaggregates (≥250 µm³) but contained a larger proportion of microaggregates (≤250 µm³) compared to the other treatments (Figure 5.2, B; One-way ANOVA, F= 550.89, P= 0.01).

Figure 5.2 I Commercial polysaccharides increase the abundance of aggregates in sandy loam soil

Wet sieving analysis used 10 mg/mL of commercial polysaccharides which were added to 1 g of sterile sandy loam soil. Commercial standards included: gum Arabic, xylan and xyloglucan; na = no addition (A). Xyloglucan was the most effective polysaccharide to increase the abundance of aggregates ≥500 µm; standard deviation bars are displayed. Asterisks indicates significant difference P= <0.05 (A). Dry dispersion analysis used 18 mm³ of sandy loam soil, which was subjected to a burst of liquid nitrogen, which projected the aggregates onto a glass slide (B-D). Commercial polysaccharides (1 µg in mL) were added to 1 mg of sterile substrate, and incubated for 2 h (B-D). Aggregates (≥500 µm³) were more abundant within soils that contained xyloglucan and xylan (C and D). Soil with gum Arabic had fewer aggregates ≥500 µm³ (B). na = no addition control (A and B); Rel. = Relative. Each data point is a mean of three biological replicates. Dashed line indicates where significance was calculated (aggregates ≥500 µm³ where compared). Soil that contained xylan and xyloglucan could significantly increase the abundance of aggregates, whereas gum Arabic did not significantly increase the abundance of aggregates, P-values shown.

5.2.3 Xyloglucan promoted aggregate formation in sandy loam soil

Scanning electron microscopy was used to visualise the effects that were previously observed when xyloglucan was added to sandy loam (Figure 5.1 and Figure 5.2, D). Xyloglucan was chosen to be imaged as it was the most effective polysaccharide to adhere to and increase the abundance of aggregates in the sandy loam soil. Xyloglucan (1 µg in 1 mL) was added to 1 mg sterile sandy loam soil, and agitated for 2 h. After agitation, soil was centrifuged with the supernatant removed. Soil was then dried for 48 h. Subsequently, soil was spread onto a glass slide with a stub containing a carbon-rich tape being dipped into the soil. A thin layer of platinum coated the particles that were stuck to the stub prior to imaging. SEM demonstrated that treating the soil with xyloglucan increased soil aggregation with small to medium sized particles (clay to slit) aggregating to each other (Figure 5.3, XG). Smaller soil particles could be seen aggregating onto the surface of the larger particles present in the sample (arrows), which has been supplemented with xyloglucan. Within the no addition control, low amounts of soil particles could be seen aggregating to larger particles (Figure 5.3, na; arrows).

Figure 5.3 I Scanning electron microscopy images of sandy loam soil with and without xyloglucan

Xyloglucan (1 µg in 1 mL) was added to 1 mg of sterile sandy loam soil, and mixed in water for 2 h. After incubation, soil was dried for 48 h before imaging. Images on top are an overview of the soil samples using a magnification of 100x. Images below focus on a representative soil particle (shown by arrows) using a magnification of 500x. Adding xyloglucan increased soil particle binding, which enhanced soil aggregate formation, na = no addition.

5.2.4 Test polysaccharide increased aggregates in ground inert glacial rock

There was an opportunity to explore the aggregation effect caused by test polysaccharides using a young soil, glacial rock which contained no organic matter. This inert glacial rock was collected by Dr Katie Field from the Fox Glacier in New Zealand. After observing an increase in the abundance of aggregates in soil, inert ground glacial rock was selected (≥500 µm dry sieved) to determine if these test polysaccharides could aggregate particles without the presence of organic matter. Test polysaccharides (10 µg in 1 mL) were added to 1 mg sterile glacial rock, and agitated for 2 h. After 2 h, glacial solutions were centrifuged with the supernatant removed. Glacial rock samples were then dried for 48 h, and assayed using dry dispersion analysis. The analysis uncovered that the test polysaccharides could increase the abundance of macroaggregates ≥500 µm³ (Figure 5.4; Mann-Whitney U, W= 224.75, P= <0.01). Xylan ranked the highest test polysaccharide to accumulate the larger aggregates of glacial rock (Figure 4.5, B; One-way ANOVA, F= 124.32, P= 0.01). This was not observed when xylan was added to sandy loam soil which contained organic matter (Figure 5.2, C).

Figure 5.4 I Commercial polysaccharides increased the abundance of aggregates of inert glacial rock

Xylan (B) was the most effective at aggregating the glacial rock ≥500 µm³ followed by xyloglucan (C) and Gum Arabic (A). Commercial polysaccharides (10 µg in 1 mL) were added to 1 mg (0.1% w/w) of sterile glacial rock, and incubated for two h. A 1:25 dilution of antigen was used; na = no substrate control. Each data point is a mean of three biological replicates. Dashed line indicates where significance as calculated (aggregates ≥500 µm³ were compared). Glacial rock that contained gum Arabic, xylan and xyloglucan could significantly increase the abundance of aggregates, P-values shown. Rel. = Relative.

5.2.5 REC1 could adhere to soil and increase the abundance of aggregates

After exploring the commercially equivalent polysaccharides released by plant roots, a sufficient amount of REC1 (~1 mg) was collected from the concentrated hydroponate of Cadenza (Chapter 4). This was in preparation for soil adherence and aggregate status analyses. REC1 was added to soil with an ELISA being undertaken to determine if REC1 could adhere to soil. A dry particle dispersion analysis also occurred to see if REC1 could aggregate soils. Fifty micrograms of REC1 was dissolved into 100 µL of dH₂O and added to 50 mg soil. Once treated soil was agitated for 2 h, with the supernatant removed and assayed using an ELISA (Figure 5.5, A). The resulting pellet was dried and analysed using dry dispersion analysis (Figure 5.5, B). The subdomains of REC1 were detected by using LM1 (extensin), LM2 (AGP), LM11 (xylan) and LM25 (xyloglucan) MAbs within an ELISA, similar to the detection of the commercial polysaccharides that were added to soil. A control, where 50 µg of REC1 was dissolved in 100 µL of dH₂O without the soil, was used.

The ELISA demonstrates that all the subdomains of REC1 could adhere to the sandy loam soil (Figure 5.5, A). The MAb signals were significantly lower compared to the no soil control (One-way Independent ANOVA, F= 6124.21, P= 0.01). For the dry dispersion analysis, the control only consisted of 100 µL dH₂O and 50 mg soil. The analysis uncovered the REC1 treatment rapidly increased the abundance of macroaggregates ≥500 µm³ compared to the no addition control (Figure 5.5, B; Two-Sample T-Test, T= 40.49, P= <0.001). The proportion of microaggregates within the REC1 treatment was significantly reduced (Figure 5.5, B; Two-Sample T-Test, T= 36.11, P= <0.05).

Figure 5.5 I REC1 could adhere to soil and increased the abundance of soil aggregates

Fifty micrograms of REC1 was added to 50 mg of sterile sandy loam soil (+), and incubated with 100 µL dH₂O for 2 h (A and B). The signals of LM1 (extensin), LM2 (AGP), LM11 (xylan), and LM25 (xyloglucan) released from the sandy loam was lower compared to the no addition control (-), which demonstrates that the components of REC1 could adhere to the sandy loam soil (A). Data are a mean of three biological replicates. A 1:125 dilution of antigen was shown; standard deviation bars are shown. Asterisks indicates significant differences, *P= 0.05, **P=0.001 and ***P= 0.0001 (A). REC1 significantly increased the abundance of aggregates (≥500 µm³) compared to the no addition control (na), P-values shown (B). The proportion of microaggregates was lower within the REC1 treatment as indicated by the arrow (B). Each data point is a mean of three biological replicates. For dry dispersion analysis only aggregates ≥500 µm³ were used to test for significance, as shown by dashed line (B). Rel. = Relative.

5.2.6 Application of REC1 resulted in more soil aggregates compared to the commercial polysaccharides

To gauge the effectiveness of REC1 at increasing the proportion of aggregates, a comparative dry dispersion analysis was undertaken using commercially equivalent polysaccharides, xylan and xyloglucan. Lower concentrations of each commercial polysaccharide were used for this assay as they were found to be more effective at aggregating soil using lower concentrations (Figure 5.4). Each polysaccharide including REC1 (50 µg) was dissolved into 100 µL of dH₂O and then added to 50 mg sterile sandy loam soil. Soil was agitated for 2 h, and centrifuged to remove the supernatants. Soil was then dried for 48 h, and examined using a dry dispersion analysis. The analysis uncovered that xylan and xyloglucan increased the abundance of aggregates ≥500 µm³ compared to the no addition (Figure 5.6). Xylan ranked the most effective commercial polysaccharide at increasing macroaggregates (Kruskal-Wallis Test H= 9.36, P= <0.01). This was also observed when xylan was added to inert glacial rock (Figure 5.4). Xylan became more effective at aggregating soil particles compared to xyloglucan when used at a lower concentration (Figure 5.2, C and D, Figure 5.6; Mann-Whitney U, W= 1962.0, P= <0.05). When REC1 was added to the soil there was also an increase in the abundance of macroaggregates ≥500 µm³ (Figure 5.6), as previously observed (Figure 5.5, B). When ranking all treatments, REC1 was the most effective aggregator of soil particles compared to commercial polysaccharides (Mann-Whitney U, W= 2608.0, P= 0.01). The portion of microaggregates was significantly less within the REC1 treatment compared to the others (Mann-Whitney U, W= 2974.0, P= 0.001). The portion of macroaggregates ≥1,000 µm³ also greatly varied within the treatments. Macroaggregates ≥1,000 µm³ represented 42% of the total soil fraction of REC1, whereas, for xylan this was 38%, and for xyloglucan this was 15% (Figure 5.6).

Figure 5.6 I REC1 was the most effective soil aggregator compared to commercial polysaccharides

Xylan, xyloglucan and REC1 (50 µg) were dissolved into 100 µL of dH₂O and added to 50 mg of sterile sandy, and incubated with for 2 h. Two no addition (na) controls were undertaken using three biological replicates each. REC1 was the most effective treatment that increased the abundance of aggregates ≥1,000 µm₃. Xylan was the second most effective treatment followed by xyloglucan. Each data point is a mean of three biological replicates; standard deviation bars are shown; asterisks (*) indicates significant difference P= <0.05, and (**) P= 0.01. Rel. = Relative.

5.2.7 REC1 promoted aggregation within sandy loam soil

To visualise the effects of adding REC1 to soil SEM was used. REC1 (50 µg) was dissolved in 100 µL of dH₂O, and then added to 50 mg of soil. This soil solution was agitated for 2 h. After agitation, soil was centrifuged with the supernatant removed. Soils were then dried for 48 h. Soil with and without REC1 were spread onto glass slides with a stub containing a carbon-rich tape being dipped into the soil, and coated with a thin layer of platinum. Overall images of the soil are placed on top panel (Figure 5.7). Representative particles chosen from both groups are placed at the bottom. There are few clay and silt particles within the no addition sample, which can be seen forming aggregates. Mostly large smooth particles can be seen within the no addition (Figure 5.7; na). This may be due to the lack of substances retaining these particles on the surface of large organic or inorganic particles. Within the soil that has had REC1, lots of aggregates made of clay and silt particles are shown, forming aggregates (Figure 5.7; REC1). A larger proportion of these aggregates are probably sticking to large organic and inorganic particles. A large group of these aggregates can also be seen within the centre of the micrograph (Figure 5.7; REC1). REC1 appears to be acting as glue, retaining these smaller particles that are present within the SEM micrographs (arrows). Within the no addition there was a low amount of smaller particles (arrows). This may have been because they had been washed out during the removal of the supernatant. This was not observed in the soil that had REC1, where small particles can be seen sticking to larger particles.

Figure 5.7 I Scanning electron microscopy images of sandy loam soil with and without REC1

REC1 (50 µg in 100 µL dH₂O) was added to 50 mg of sterile sandy loam soil, and incubated for 2 h. After incubation, soil was dried for 48 h prior to imaging. Images on top are an overview of the soil samples using a magnification of 70x. Images below focus on a representative soil particle using a magnification of 300x. Adding REC1 to soil increased soil aggregation, which in turn increased the appearance of soil particles that were rough denoted by arrows. Low amounts of small particles (arrow) can be seen within the no addition (na), which may have been lost during the removal of the supernatant prior to drying. Small clay particles can be seen clinging onto larger silt particles (arrows).

5.2.8 Immunofluorescence microscopy of sandy loam soil reveals xyloglucan adhering to soil particles

To visualise the locations where polysaccharides were binding to soil particles, immunofluorescence microscopy was used. Commercial polysaccharides, xylan and xyloglucan, were selected for immunofluorescence microscopy as there were MAbs which could be used to reveal where they were binding. This would not have been possible for REC1 as there are no MAbs raised against this multi-polysaccharide complex. Although four MAbs can be used to infer the presence REC1 this would result in more of the preparation being used for imaging particularly, when REC1 is in such short supply. Xyloglucan (10 µg in 1 mL) was added to 1 mg of soil, and agitated for 2 h. Once agitated, soil was centrifuged with the supernatant removed, and the pellet dried for 48 h. After drying, soil was placed into eight well glass slides that had been Vectabonded. Soil was then probed with LM25, and stained with anti-rat-FITC to visualise xyloglucan binding. Soil was imaged using a 40x objective. When xyloglucan was added to the soil, bright fluorescence was clearly shown where aggregates were present. This was demonstrated when the bright-field and FITC channels were combined (Figure 5.8; +LM25, +XG). When combined the bright-field and FITC channels there was no florescence when the xyloglucan was excluded from the soil (Figure 5.8; +LM25, -XG), and when LM25 was excluded from the soil (Figure 5.8; -LM25, +XG).

Figure 5.8 I Detection of xyloglucan that was added to sterile sandy loam soil

Xyloglucan was added at 10 µg in 1 mL to 1 mg of sterile sandy loam soil, and incubated for 2 h. Sandy loam soil then was centrifuged, and supernatant removed. Soil was then added to an eight well Vectabonded slide. Signals of LM25 can be clearly seen where soil aggregates were present. When removing either xyloglucan or LM25 from the soil no fluorescence was shown. Toluidine Blue O (0.1%; pH 5.5 0.2 M phosphate) was added to each well to remove autofluorescence. Scale bar = 50 µm.

5.2.9 Immunofluorescence microscopy of soil reveals xylan adhering to soil particles

To demonstrate that xylan could also be seen adhering to soil aggregates, immunofluorescence microscopy was undertaken. Xylan (10 µg in 1 mL) was added to 1 mg of soil, and aggregated for 2 h. Once agitated, soil was centrifuged with the supernatant removed, and the pellet dried for 48 h. After drying, soil was placed into eight well glass slides that had been coated with Vectabonded adhesive agent. Soil was then probed with LM11, and stained with anti-rat-FITC to visualise xylan binding. Soil was imaged using a 40x objective. When xylan was added to the soil, strong fluorescence was apparent where the aggregates were present. This was demonstrated when the bright-field and FITC channels were combined (Figure 5.9; +LM11, +X). When combining the bright-field and FITC channels, there was no florescence when the xylan was excluded from the soil (Figure 5.9; +LM11, -X), and when LM11 was excluded from the soil (Figure 5.9; -LM11, +X).

Figure 5.9 I Detection of xylan that was added to a sterile sandy loam

Xylan was added at 10 µg (in 1 mL) to 1 mg of sterile sandy loam soil, and incubated for 2 h. Sandy loam soil was centrifuged, and supernatant removed. Soil was then added to an eight well Vectabonded slide. Signals of LM11 were clear where soil aggregates were present. When removing either xylan or LM11 from the soil no fluorescence was shown. Toluidine Blue O (0.1%; pH 5.5 0.2 M phosphate) was added to each well to remove autofluorescence. Scale bar = 50 µm.

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5.3 Discussion

5.3.1 Soil aggregation enhances root-soil contact

This research has shed a new light in the involvement of plant-derived polysaccharides in soil aggregate status. The current view of polysaccharides and glycoproteins released from plant roots is that they solely serve to lubricate roots to reduce friction as they penetrate through deeper layers of soil (Guinel and McCully 1986; Read and Gregory 1997). Research using isolated root mucilage from maize has hinted at the involvement of polysaccharide release by roots in aggregate status (Tisdall and Oades 1982; Morel at al. 1991; Watt et al. 1993). However, no detailed analysis of plant-derived polysaccharides involved in soil aggregate formation has been carried out.

This investigation has taken commercial plant-derived polysaccharides, xylan and xyloglucan, and has determined that they increase the abundance of aggregates within soil (Figure 5.2). Furthermore, these commercial polysaccharides increased the abundance of aggregates within glacial rock, without the presence of native organic residue (Figure 5.4). The abundance of soil aggregates had also rapidly increased when an isolated sample of Root Exudate Complex 1 (REC1) (Chapter 4) from the hydroponate of Cadenza was added to soil (Figure 5.5). When REC1 was compared to the commercial polysaccharides, REC1 was the most effective aggregator of soil (Figure 5.6). Increasing the abundance of aggregates enhances the rhizosheath, which in turns increases the extraction of water and nutrients from the soil, and provides a superior platform for initiating symbiotic associations with mycorrhizal fungi and nitrogen-fixing bacteria, and increases water infiltration capacity and soil aeration (Hartnett et al. 2012; Lehmann and Kleber 2015).

5.3.2 Plant-based polysaccharides cause soil aggregation

For the first time, immunofluorescence staining has been utilised on soil to enable the visualisation of xylan (Figure 5.8) and xyloglucan that was added to soil (Figure 5.9). When stained, both commercial polysaccharides were only located to aggregates. Previous studies were unable to visualise the binding of plant-derived polysaccharides within soils due to a lack of molecular probes (Oades 1978; Tisdall and Oades 1982; Oades 1984; De Booth et al. 1984; Cheshire 1990). Only the resulting effects of the polysaccharides could be documented using light or electron microscopy. This investigation determined that commercial plant-derived polysaccharides added to soil had adhered differently to different soil types (Figure 5.1). It has been documented that polysaccharides initially adhere to the smallest soil particles, clay, to form microaggregates (Day 1965; Oades 1984; McCully 1999). Once microaggregates (≤250 µm³) form they can also bind to each other to form macroaggregates (≥250 µm³). As the amount of clay particles varies between soil types (Brown 2008), the binding of commercial polysaccharide would inevitably vary. The fewer clay particles available, the less a polysaccharide could bind. Clay loam, the substrate with the most clay particles, had the largest reduction of commercial polysaccharides detected within the supernatant (Figure 5.1). As sand contains no clay particles there should be no binding. However, there was a significant reduction in the commercial polysaccharides added.

The amount of polysaccharide added to the substrate was 1% (w/w), which may have saturated the substrate with commercial polysaccharide. The amount added may seem credibly high, and not presentative of a native soil, however, research suggests levels of polysaccharide released would be within this concentration close to the root cap and tips of plants (Oades 1984; Morel et al. 1991; Watt et al. 1993; Traore et al. 2000). Previous research adding commercial polysaccharides to soil, xanthan (Chang et al. 2015), polygalacturonic acid and glucose (Tisdall and Oades 1982; May et al. 1993) and guard gum (Metha et al. 1960) used concentrations of 1% (w/w), similar to this investigation (Figure 5.2). These studies also revealed that the commercial polysaccharides could aggregate soil particles. However, these studies did not investigate the stability of these aggregates.

It has yet to be determined if plant roots can regulate the rhizosphere depending on the needs of the plant for instance, to aid with water uptake during periods of drought. However, some research examining the rhizosheaths of various grasses, including spotted brachiaria (Brachiaria nigropedata) and canegrass (Eragrostis rigidior), has indicated that these plants could alter the thickness of their rhizosheaths in response to drought (Hartnett et al. 2012). Moreover, there were differences within the thickness of the rhizosheath between the subfamilies of Poaceae during drought. For instance, Arundinoideae (C3 grasses; including giant reed) had a significantly thicker rhizosheaths with a mean diameter of ~529 µm compared to Panicoideae (including maize and sugar cane), which had a mean diameter of ~280 µm (Hartnett et al. 2012). It may be possible that other grasses including cereals (particularly wheat) may form different rhizosheaths that could be strengthened during periods of drought. To date, no research has been undertaken to explore the temporal dynamics of the rhizosheath. Plants may even a high burst of polysaccharides as a part of their attempts to establish their root network. Once established, plants may reduce the release of these polysaccharides as they begin to establish their rhizosheath.

5.3.3 Aggregates can be formed from plant-derived polysaccharides

Isolated maize root mucilage (Oades 1984; Watt et al. 1993; McCully and Sealey 1996) or modelled mucilages using polygalacturonic acid and glucose (Tisdall and Oades 1982; May et al. 1993) were the only examples of plant-based polysaccharides that were shown to be involved in the formation of soil aggregates. This limited use of plant-based polysaccharides had left a wide gap in knowledge. The only well documented example of a polysaccharide released by an organism, which caused soil to aggregate is xanthan gum from the bacteria Xanthomonas campestri (Chang et al. 2015). This study uncovered that the exuded polysaccharides from Xanthomonas acted in a similar way to that of modelled root mucilage and root mucilage isolated from maize. Microorganisms in soils are known to release exopolysaccharides in order to bind together, forming an extracellular matrix (Griffiths and Jones 1965; Chang et al. 2015). This matrix aids biochemical reactions between the cells, supports molecular signalling and resource shuttling (Flemming and Wingender 2010). As an indirect function, these exopolysaccharides enhance the interface that these organisms have with the soil (Flemming and Wingender 2010). This interface increases uptake of water and nutrients to cells just like the rhizosheath. Perhaps, the majority of soil dwelling organisms release polysaccharides either directly or indirectly to maintain this life supporting contact with the soil.

Wet sieving and dry particle dispersion analyses revealed that aggregates formed from commercial polysaccharides were highly stable (Figure 5.2). Subjecting these aggregates, that had been hydrated, with a high flow of water, and high mechanical pressure did not disrupt these aggregates. The abundance of macroaggregates ≥500 µm³ was typically observed when the commercial polysaccharides, xylan and xyloglucan as well as REC1 were added to soil (each at 0.1% w/w). Furthermore, polygalacturonic acid, a commercial form of pectin, was also found to increase the abundance of macroaggregates ≥500 µm³ in soil but to a lesser amount as to xylan and xyloglucan (data not included). These increases in aggregate size are twice the size of previously determined aggregates (typically ≤250 µm³) formed from modelled root mucilage, polygalacturonic acid and glucose, and isolated maize root mucilage (Tisdall and Oades 1982, May et al. 1993). The accumulation of macroaggregates due to the commercial polysaccharides and REC1 has until now only been reported to be caused by roots, hyphae networks and decaying organic matter such as leaves (Reid and Goss 1981; Tisdall 1994).

When using SEM, the binding of small particles to larger ones, producing aggregates was observed when xyloglucan and REC1 was added to soil (Figure 5.3). The sizes of the aggregates observed within SEM matched what was being observed within the aggregate stability assays. This supports previous research, which determined that soil aggregates formed by polysaccharides derived from decaying leaf matter are highly stable (Cheshire et al. 1985; Cheshire 1990). Aggregates formed by these polysaccharides could only be disrupted using sodium periodate which oxidises the bonds between adjacent carboxyl and hydroxyl-bearing carbons within the polysaccharide, which in turn degrades the polysaccharide strands holding clay particles together (Clapp and Emerson 1965; Cheshire et al. 1985). It would be of interest to explore the effects on soil structure and aggregate stability using a larger library of commercially available plant-derived polysaccharides. Perhaps, with more research a soil conditioner using a more cost effective form of commercial polysaccharide or with a blended combination of commercial polysaccharides could be developed to maintain or enhance soil aggregates. This product could help to prevent soil erosion, which is a major problem caused modern-day agricultural production.

5.3.4 Aggregation formation in soil is highly dynamic

Polysaccharides are thought to cause aggregation by binding to clay particles through weak hydrogen bonds between the carboxyl, and to a lesser degree the hydroxyl groups present on the molecule, to the cations attached to the surface of the clay particle (Foster 1981, Traore et al. 2000; Olsson et al. 2011; Figure 1.4). Once polysaccharides become bound to the surface of clay particles they form bridges between other clay particles, thus causing soil particles to form microaggregates (≤250 µm³; Foster 1981, Fitz Patrick 1993; Traore et al. 2000). These microaggregates then bind together to form macroaggregates (≥250 µm³; Tisdall and Oades 1982; Cheshire 1990). Inert glacial rock was also assayed within this study, which revealed a comparable aggregation effect to that observed using sterile sandy loam soil (Figure 5.4). This supports the hypothesis of polysaccharide adhering to the inert surface of clay particles through cations, such as Ca²⁺, K⁺ and Mg²⁺. If aggregation only occurred within sterile sandy loam soil, it would suggest that binding only occurred with the already present organic debris. Perhaps polysaccharides can adhere to both the organic and inorganic particles in soil. Previous work indicates that when polysaccharides are bound to clay particles, the molecules are inaccessible to soil dwelling microorganisms (Tisdall and Oades 1982; Cheshire 1990). It would be interesting to undertake xylanase and xyloglucanase treatments of soil and inert glacial rock with the addition of xylan and xyloglucan. Preliminary work suggests that these polysaccharides cannot be degraded by enzymes when bound to soil particles (Cheshire 1990). This adds to the growing body of evidence that these commercial polysaccharides form highly stable aggregates.

Polysaccharide aggregates are not held together indefinitely but degrade over time due to gradual degradation of the polysaccharides (De Booth et al. 1984). A continual release is, therefore, required to maintain the stability of these aggregates within the rhizosphere, which may account for the continual detection of release cell wall-related molecules (Greenland 1979; Tisdall and Oades 1982; Cheshire 1990). Moreover, if certain nutrients including Fe³⁺ become limited, roots can exude chelating agents. These agents target the ions bound to clay particles, which can disrupt the polysaccharide-clay particle binding (De Booth et al. 1984; McNear 2013). Once plants receive sufficient ions, roots stop exuding these agents. When sufficient ions have replaced the exposed surface of clay, released polysaccharides can then bind to the clay. This demonstrates that the turnover of aggregates is highly dynamic, and that plants have a rudimental control over aggregate turnover. Growing crops in nutrient deprived soils or by including heavy metals could help further explore this aggregate turnover.

Conditions that are optimum for the highest capacity for polysaccharide-clay binding are fertile soils with acidic polysaccharides (Cheshire 1979; Cheshire et al. 1979; Cheshire 1990). Acidic polysaccharides are generally more ionised compared to their neutral counterparts, making them possess greater anionic electrolyte properties (Edwards and Bremner 1967; Cheshire 1990). Neutral polysaccharides typically secreted from bacteria have a weaker capacity for soil aggregation (Clapp and Davis 1970). Releasing an acidic molecule into the soil would, therefore, be favourable to enhance soil aggregation, and the stability of these aggregates. As REC1 is potential acidic multi-polysaccharide complex (Chapter 4; Figure 4.3), released by wheat, barley and maize, it would support this hypothesis. This also suggests that the AGP core of REC1 may be acting not just as a crosslinker for the other subdomains, but as a key determinate of soil binding. AGP may act as a multi-directional bridge between clay particles with the subdomains. It would be interesting to verify this multi-directional bridge hypothesis by using a purified sample of APAP1 (Tan et al. 2013), which also has an AGP core. As gum Arabic, a commercial form of AGP, was ineffective at increasing aggregate size during water saturation, and was least effective at increasing aggregate size during mechanical pressure. This supports the hypothesis that AGP is acting as a multi-directional bridge, which does not directly support polysaccharide-clay binding, but by itself AGP is a poor soil aggregator. This may explain the possible reason for the other subdomains of REC1. More research is required to explore this hypothesis. The stability of aggregates caused by polysaccharide is believed to be directly proportional to the molecular weight of the molecule binding them together (Cheshire 1990). This further supports why REC1 was the most effective soil aggregator, as REC1 is a putative multi-polysaccharide complex, having between four and five domains it is hypothesized to have a high molecular weight.

A common practice amongst farmers is to plough fields so that they can suppress weed growth, which hinders plant growth, prepare fields for seed planting and to incorporate fertiliser (Klute 1982). However, tillage, the process of agitating soil, breakdowns these micro- and macroaggregates that have been formed whilst crops were growing (Six and Paustian 1999; Ji et al. 2013). This not just disrupts the rhizosheath but other soil contact that soil-dwelling organisms have developed, thus eroding the soil. It has been demonstrated that restoring these aggregates within eroded soils can recover soil water infiltration capacity and aeration (Erktan et al. 2016). Polysaccharides derived from plants and other microbiota could be investigated for commercial use as a soil conditioner to reducing soil erosion due to tillage.

5.3.5 Released polysaccharides may have multiple roles

It is reasonable to suggest that released polysaccharides serve other purposes than root cap lubrication and soil aggregation. These polysaccharides could be used to cultivate microorganisms for plant growth. Several studies have used isolated root mucilage exuded by pea and alfalfa (May et al. 1993; Knee et al. 2001; Gunina and Kuzyakov 2015; Sun et al. 2015). These root mucilages were placed into agar, which had been inoculated with rhziobacteria. These bacteria proliferated in the presence of these high molecular weight compounds. Colonies of soil bacteria could also proliferate in the presence of isolated maize root mucilage (Knee et al. 2001; Walker et al. 2003); this was also shown for infectious bacteria (Zheng et al. 2015). Arbuscular mycorrhizal fungi have also been shown to contain plant-specific degrading enzymes, such as xyloglucanase (Rejon-Palomares et al. 1996). Evidence for polysaccharide catabolism of plant high molecular weight compounds in mycorrhizal fungi remains unclear. It may be possible that plants have some regulatory control over the amount, and which polysaccharides that they release. More research is required to explore how microorganisms use released polysaccharides for growth, and if a plant has regulatory control over their secretion.

5.3.6 Soil aggregation may have been vital for land colonisation

Without soil, most land plants would not be able to survive. The development of this medium was crucial for the development of complex plants that are present within many ecosystems, which are utilised for food, pharma and fabric production (Brown 2008; Prosser 2015). Prior to the colonisation of land plants, primitive soils would be comparable to glacial rock deposits that were formed by weathering processes (Huggett 1998). As early plants began to take the initial foot steps to complexity, over 470 million years ago (Kenrick and Crane 1997; Bateman et al. 1998; Popper et al. 2011), they had to develop a relationship with early soils. Early plants had to remove water and necessary nutrients from the inert rock minerals. Aggregating these inert minerals together could have developed a primitive rhizosheath as well as to anchor these early land plants (Huggett 1998; Konrat et al. 2008; Prosser 2015).

REC2 is an acidic macromolecule due to the presence of AGP, and acidic xyloglucan (Peña et al. 2008). This acidity would have maximised the binding of micro-particles within the early soils (Tisdall and Oades 1982; Chenu and Guerif 1989; Cheshire 1990). As well as being acidic, the molecular weight of REC2 would have contributed to the binding of this multi-polysaccharide complex to inert minerals. More research is required to explore the effectiveness of REC2 to aggregate inert glacial rock. If aggregation occurs, it would support this hypothesis. As plants became more complex, comparable to today’s cultivars, this released macromolecule may have developed more subdomains to increase the effectiveness of soil aggregation.

The development of a released multi-polysaccharide complex may have corresponded to the ever increasing complexity of soil. Plants may have further added more polysaccharide subdomains to increase the binding sites used in clay binding (Olness and Clapp 1973; Chenu and Guerif 1989). The appearance of early land plants corresponds to the development of xyloglucan (Popper and Fry 2003; Popper et al. 2011), which was acidic (Peña et al. 2008). Perhaps, AGP-xyloglucan (REC2 subdomains) was the most effective soil aggregator until AGP-xyloglucan-extensin-xylan (REC1 subdomains) evolved, superseding AGP-xyloglucan as the most effective soil aggregator. As more and more generations of plants colonised the land, a continual amount of organic matter was deposited into the evolving soil through decaying plant matter (Mitchell et al. 2016; Rimington et al. 2016). This matter would have greatly contributed to the development of macroaggregates, and soils (Kenrick and Crane 1997; Konrat et al. 2008). However, the ability to retain and maintain these aggregates surrounding roots within the rhizosphere would have been vital for safeguarding the root-soil interface. It would also be interesting to determine when plants evolved this ability to form a rhizosphere.

5.4 Conclusion

The rhizosheath serves as a vital interface for plants to extract water and nutrients from the soil, serve as a means of anchorage, and to develop symbiotic relationships with soil dwelling microorganisms. Commercial plant-derived polysaccharides, xylan and xyloglucan could readily bind to particles within various soil types that are commonplace throughout many ecosystems. After the commercial polysaccharides adhered to soil they increased the abundance of aggregates within sandy loam soil and glacial rock. Aggregates formed as a result of commercial polysaccharides, remained stable during high water stress, and high mechanical pressure testing. Given more research, a commercial polysaccharide or blend of commercial polysaccharides could be developed to produce a soil conditioner. This conditioner could be used to fight against soil erosion. An isolated sample of REC1 was added to soil, which rapidly increased the abundance of macroaggregates during high mechanical pressure. The AGP within REC1 may act like a multi-directional bridge, with the subdomains binding to a large volume of clay particles. This would make REC1 an advanced aggregator compared to the commercial polysaccharides tested. A similar complex to REC1, REC2, was uncovered to be released from the basal plant liverwort. This finding suggests that these released multi-polysaccharide complexes may have played an important function in the initial formation of the rhizosheath, and modern-day soils.

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