Characterisation of polysaccharides released from the roots of hydroponically grown crops

3.1 Introduction

Little is known about the identity of polysaccharides released from plant roots and their function. MAbs are highly efficient and sensitive molecular probes that can be used to detect epitopes of polysaccharides (Lee et al. 2011). Physio-chemical methods of identifying polysaccharides are time consuming, and usually require a large amount of material which has been purified, for instance, a minimum of ~1 mg is required for monosaccharide linkage analysis (Moller et al. 2008; Pattathil et al. 2012). Whereas, immuno-based methods such as ELISA require approximately 100-fold less material, which does not require as much purification (Lee et al. 2011) to reveal the epitopes of cell wall polysaccharides. Previous attempts that have isolated polysaccharides released from roots (Chaboud and Rougier 1984; Bacic et al. 1986; Moody et al. 1988; Osborn et al. 1999) produced little material (up to tens of micrograms) for physio-chemical analysis. Prior to this investigation, no studies have used an immuno-based approach to screen the root exudate for the epitopes of cell wall polysaccharides released by plant roots. There is a large library of MAbs raised against a range of cell wall polysaccharides, and which may aid in deciphering the complexity of polysaccharides released from plant roots. Although MAbs are efficient, they may not identify all the polysaccharides that are released by plant roots. If there is a polysaccharide released by plant roots that does not contain epitopes that the current MAb library recognise, then it will not be detected. Whereas, for a more physio-chemical approach, such as monosaccharide composition and monosaccharide linkage analysis, can identify all polysaccharides present.

This investigation focused on growing plants hydroponically so that the polysaccharides released from roots could be efficiently isolated for immunochemical and biochemical analysis. Furthermore, by growing plants hydroponically it could be assured that the polysaccharides detected originated from the root network, and not from the seed coats or leaves. This study has also selected to grow a range of crop species, wheat, barley, maize, pea, tomato and rapeseed, which develop large root networks, maximising the probability of collecting sufficient material for biochemical analysis.

The first aim of this project was to identify the major cell wall polysaccharide epitopes released into the hydroponic medium of various crop species using MAbs, with a particular focus on three wheat cultivars, Avalon, Cadenza and Skyfall. Focusing on three cultivars of wheat can determine if there is a genetic factor that is involved in the release of polysaccharide through any differences observed between the polysaccharides detected in their growth medium. By using MAbs in conjunction with ELISA, the epitopes released by wheat roots were determined, and compared in the form of epitope profiles. From this initial exploration, these epitope profiles were compared to the cell walls of the root body of the same wheat cultivar screened. Cell wall polysaccharide epitope profiles from three major eudicotyledon crops, pea, tomato and rapeseed, and two other major cereal crops, barley and maize, were then compared.

3.2 Results

3.2.1 There were no differences in root growth between the wheat cultivars

To assess the effects of the hydroponic system on the growth of wheat, the root lengths and root fresh weights of three wheat (Triticum aestivum) cultivars, Avalon, Cadenza and Skyfall were compared. These cultivars represent a range of wheat varieties, Avalon and Skyfall are both widely grown winter varieties whereas, Cadenza is an older and less popular spring variety (Kumar et al. 2011). Prior to hydroponics, wheat were germinated in vermiculite and perlite (50:50) for one week in a growth cabinet until, they were strong enough to be transferred to the hydroponic system. Once in the hydroponic system wheat were grown for an additional two weeks. Two weeks was found to be an effective period to allow polysaccharides to accumulate in the hydroponates to be readily detected. After two weeks of hydroponics, the hydroponates of the wheat cultivars were each filtered using Whatman filter paper to remove any plant debris and then concentrated (from 9 L to ~200 mL). Once a bucket had been filtered the wheat were harvested with their longest root lengths and total fresh root weights measured. After measuring, it was determined that the longest root lengths of the cultivars were not significantly different (One-Way Independent ANOVA, F= 0.67, P= <0.05), being on average 45 cm long (Figure 3.1, A). The total fresh root weights were also not significantly different (One-Way Independent ANOVA, F= 0.87, P= 0.544), being on average 4.42 g (Figure 3.1, B). Overall, the growth of the three wheat cultivars was not statistically different.

Figure 3.1 I Mean root lengths and root fresh weights of the wheat cultivars

After two weeks of hydroponics, the longest root lengths and total fresh weights were recorded. There was no significant difference between the mean longest root length and total fresh weights of the wheat cultivars, Avalon, Cadenza and Skyfall. Twelve wheats were grown in one 9 L bucket to form one biological replicate. A total of three biological replicates were used. Standard deviation bars shown.

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3.2.2 Wheat roots release AGP, extensin, xylan and xyloglucan epitopes

To reveal the identity of the polysaccharide epitopes released by wheat roots, the hydroponates of wheat were probed with a range of MAbs. After two weeks of hydroponics, plants were harvested, and the hydroponate directly assayed using an ELISA. An array of 29 MAbs was used to probe the hydroponate to reveal the types of cell wall-related polysaccharides being released by the roots of the wheat cultivars (Table 2.1). The resulting heat map revealed that a diverse mixture of polysaccharide epitopes were present within each of the hydroponates of the wheat cultivars (Table 3.1). The top ranking MAb signals were epitopes of xyloglucan, xylan, extensin and AGP. Unexpectedly, signals from the epitopes of pectic-related polysaccharides were either very low or not present across the cultivars (Table 3.1). An interesting observation was made when LM8 (XGA) was not detected within the hydroponate of the cultivars. The LM8 epitope is specific to root cap cells of a range of angiosperm including pea and maize (Willats et al. 2004).

LM2 had the highest signal from the AGP-specific MAbs across the cultivars. This indicates that the AGP has a b-linked glucuronic acid residues (Table 3.1). The signals of MAbs, which were specific to one polysaccharide varied, suggesting that there may be different forms of those polysaccharides being released from each cultivar. For example, LM25 was the only signal from a xyloglucan-binding MAb that was present in the hydroponate of Cadenza. Within the hydroponates of Avalon and Skyfall, there was a clear signal from LM15, suggesting that the xyloglucan may be less galactosylated compared to Cadenza (Table 3.1).

Table 3.1 I Heat map of polysaccharide epitopes released from the roots of wheat cultivars, Cadenza, Avalon and Skyfall as determined by ELISA

Twenty-nine monoclonal antibodies raised against cell wall polysaccharides were used to screen the hydroponates of three-week-old wheat cultivars through ELISA. Form the heat map, MAb signals from LM2 (AGP), LM1 (extensin), LM11 (xylan) and LM25 (xyloglucan) were ranked the highest across the wheat cultivars. An aliquot (9 mL) from each bucket was removed for one replicate, 1 mL 10x PBS was then added to each aliquot for the ELISA. The 1:25 dilution of the hydroponate was used for this analysis. Each data point is a mean of three biological replicates. Twelve wheat plants were grown per cultivar per replicate. A cut off point of 0.1 au was removed, any values below this level were regarded as having no signal (-), shading of heat map was across the cultivars screened.

3.2.3 A 30 KDa cut-off point membrane was suitable to concentrate polysaccharides

In order to explore the polysaccharides released by roots, the hydroponic media were concentrated from 9 L volumes to a more manageable volume (~200 mL). In order to determine which membrane size would be used for ultrafiltration, aliquots of wheat cv. Cadenza hydroponate were pipetted into a range of molecular weight cut-off point centrifuge tubes (10 KDa, 30 KDa, 50 KDa and 100 KDa). After centrifugation, the filtrate was removed. Aliquots of deionised water were placed on top of the filter, mixed and the retentate removed. Both the filtrate and retentate were assayed using an ELISA. LM25 was used as a marker to assay the hydroponate due to having the highest signal (Table 3.1).

When the hydroponate was passed through the 100 KDa and 50 KDa membranes, the LM25 signal was not retained by the membrane. When the hydroponate was passed through the 30 KDa membrane, the LM25 signal was retained (Figure 3.2). When using the larger 30 KDa cut-off point membrane within the ultrafiltration system, the MAbs with the highest signals (LM1, LM2, LM11 and LM25; Table 3.1) were used to assay the retentate and filtrate of the hydroponate. The signals from these MAbs were only present within the retentate, confirming that the 30 KDa cut-off point filter was retaining the high molecular weight compounds present within the hydroponate (Figure 3.3). This demonstrates that the 30 KDa membrane would retain four major polysaccharide epitopes within the hydroponate, and therefore, would be used to concentrate the high molecular weight compounds within the hydroponate.

Figure 3.2 I Determining the molecular weight cut-off point to use to concentrate the released polysaccharides within the hydroponate

Aliquots of wheat cv. Cadenza hydroponate (1 mL) were added to a range of 2 mL Vivaspin tubes with the following cut-off points, 100 KDa, 50 KDa, 30 KDa and 10 KDa. Tubes were centrifuged for 5 mins at 3,893 x g. The resulting filtrate was then collected. Aliquots (1 mL) of dH₂O were then used to wash the filters to remove retentate. Both the filtrate and retentate were assayed using ELISA. As LM25 (xyloglucan) had the highest signals within the hydroponates of wheat, it was used to explore which membrane cut-off point to use for concentration. The signal of LM25 was retained when using the 30 KDa cut-off point. The filtrate fraction of the 30 KDa membrane contained no detectable amounts of xyloglucan epitopes, whereas, the retentate fraction contained high amounts of xyloglucan epitopes. Standard deviation bars are shown; asterisk indicates significant difference P= <0.05.

3.2.4 The four major epitopes were still present within the concentrated hydroponate of wheat cultivars

To determine the effects of concentrating the hydroponate on the polysaccharide epitopes, the major epitopes were re-assayed. Each bucket of hydroponate was filtered using Whatman paper filter to remove any particulates. Hydroponates were then concentrated (9 L to ~200 mL) using an ultrafiltration system that used a 30 KDa cut-off point membrane. After concentrating, the hydroponates were dialysed in dH₂O with six changes, and then freeze-dried. Known quantities (10 µg/mL) were re-suspended in 1x PBS, and assayed using an ELISA. To confirm that the concentrated hydroponate retained the major epitopes, LM1, LM2, LM11 and LM25 were used to probe the newly concentrated hydroponates of the wheat cultivars. A similar profile of the major epitopes was evident (Table 3.1 and Figure 3.3); LM25 had the highest signals followed by LM11, LM2 and LM1. This demonstrated that by concentrating the hydroponate the four majority polysaccharides were being retained (Figure 3.3).

Figure 3.3 I The four major polysaccharide epitopes detected within the hydroponate of the wheat cultivars were retained after concentrating

A similar profile of released epitopes was detected after concentrating 9 L of hydroponate to ~200 mL. After concentrating, hydroponates were dialysed in dH2O with six changes, and then freeze-dried. Quantities of total dried hydroponate that was above 30 KDa (10 µg/mL) were re-suspended in 1x PBS for assaying. MAb signals of LM25 (xyloglucan) were the highest across the cultivars followed by LM11 (xylan), LM1 (extensin) and LM2 (AGP). Overall Skyfall had the lowest MAb signals when compared to the other cultivars. Data are a mean of three biological replicates. Twelve plants were grown per bucket, per cultivar, per replicate. Error bars indicate standard deviation.

3.2.5 Cell wall polysaccharide profile of released polysaccharides differs to the root body

After examining the released polysaccharides within the directly sampled and concentrated hydroponates of wheat, the cell wall polysaccharides of the root body were extracted. The resulting relative epitope levels of the root body were compared to the concentrated hydroponate. Cadenza was selected for further analysis due to the hydroponate of the cultivar containing the highest signals of the MAbs screened (Table 3.1). Once Cadenza had been grown hydroponically for two weeks, the roots were harvested, frozen in liquid nitrogen and freeze dried. Subsequently, the root material was homogenised and underwent a sequential series of dehydration steps. Once a sample of AIR was attained the material was extracted using 4 M KOH and 1% NaBH₄. The four major polysaccharide epitopes detected in the hydroponate of wheat, AGP, extensin, xylan and xyloglucan (Table 3.1) were used for this analysis. The epitope profiles of extensin (LM1) and xyloglucan (LM25) were higher within the hydroponate compared with the root body (Figure 3.4). The epitope profile of AGP (LM2) was similar in the hydroponate and the root body. However, the epitope profile of xylan was higher within the root body compared to the hydroponate (Figure 3.4).

Figure 3.4 I ELISA analysis of the root body and concentrated hydroponate of Cadenza

Wheat cv. Cadenza was grown hydroponically for two weeks. After two weeks, one wheat was taken from each bucket with the roots being cut from the stem and leaves. Roots were then frozen in liquid nitrogen and freeze dried. After drying, roots were homogenised with 10 mg of material being dehydrated using a sequential step of EtOH, acetone and methanol:chloroform. After dehydration, 1 mg of material was extracted using 4 M KOH and 1% NaBH₄. The epitope profiles of LM1 (extensin) and LM25 (xyloglucan) were higher within the hydroponate of wheat compared to the root body. However, the epitope profile of LM11 (xylan) was higher within the root body compared to the hydroponate. The epitope profile of LM2 (AGP) was similar when comparing the hydroponate with the root body. The 1:125 dilution was used for the analysis. Data are a mean of three biological replicates; one root system was selected per bucket. Relative ELISA absorbance values were determined by measuring the absorbance at 450 nm. Standard deviation bars are shown.

3.2.6 Cereal crops release relatively more xyloglucan compared to eudicotyledons

To expand knowledge of cell wall epitopes released by roots, epitope profiles of wheat were compared to five additional crop species, which were also grown hydroponically, barley (Hordeum vulgare cv. Golden Primrose), maize (Zea mays F1 cv. Earlibird), pea (Pisum sativum cv. Avola), tomato (Solanum lycopersicum cv. Ailsa Craig) and rapeseed (Brassica napus cv. Extrovert). These species represent a range of plant types including other cereals (barley and maize), Fabaceae or legumes (pea), Solananceae (tomato), and Brassica (rapeseed) which is a relative of Arabidopsis. Additionally, each of these species was found to have produced a strong root system within the hydroponic system chosen.

Each crop was germinated in 50:50 mixture of vermiculite and perlite for one week before being transferred to the hydroponics system. After two weeks, hydroponates were directly sampled and assayed using an ELISA. Two weeks was also found to be an effective period to allow polysaccharides to accumulate in the hydroponates of the species to be readily detected. The MAbs demonstrated that each crop released a unique profile of epitopes. Across the species, the relative epitopes of xyloglucan, extensin, xylan and AGP were present within the hydroponates screened (Table 3.2). For barley and maize, LM25 dominated the xyloglucan-binding MAb signals (Table 3.2). The only detectable AGP-related MAb relative signal within the hydroponates of barley and maize was LM2. This indicates that the AGP released by the roots of barley and maize contained b-linked glucuronic acid. Within the hydroponate of barley, LM11 was the only signal from the xylan-specific MAbs, indicating the presence of heteroxylan. For maize, there were signals from LM11 and LM12. The binding of LM12 within the hydroponate of indicates that the xylan released by the roots of maize is possibly feruloylated (Table 3.2; Pedersen et al. 2012). A signal from LM28, which recognises the epitopes of glucuronosyl substituted xylans, was also present in barley and maize hydroponates. Barley hydroponate had a high a signal of LM6, which indicated the present of RG-I (arabinan) or an AGP glycan (Table 3.2; Willats et al. 1998a).

The hydroponates of the eudicotyledons, pea, tomato and rapeseed, crops had relatively lower signals of LM25 compared to the cereals (Table 3.2). The highest xyloglucan-specific MAb signal was from LM25. Relatively weak signals of LM11 were detected across the eudicotyledons, indicating that they released low amounts of heteroxylan epitopes. The hydroponate of tomato had the highest relative signal of LM2, indicating the presence of b-linked glucuronic acid AGP epitopes. Whereas, for pea and rapeseed MAC207 had the highest signal, suggesting that they released different types of epitopes on the arabinogalactan domain present within AGP (Smallwood et al. 1996; Yates et al. 1996). Generally, there were higher signals from pectic and heteromannan MAbs within the eudicotyledon hydroponates compared to the cereal hydroponates. In particular, tomato had a high signal from JIM7 that binds to the epitopes of pectin (HG; Clausen et al. 2003), and LM6 that recognises the epitopes of RG-I (arabinan; Willats et al. 1998a; Table 3.2).

Table 3.2 I Heat map of polysaccharides released from the roots of barley, maize, pea, tomato and rapeseed as determined by ELISA

Twenty-nine cell wall monoclonal antibodies were used to screen the hydroponates of three-week-old barley, maize (F1 cv. Earlibird), pea, tomato and rapeseed through ELISA. From the heat map of the hydroponates, each species released a unique epitope profile. The relative epitope levels of AGP (LM2), extensin (LM1 and JIM20), xylan (LM11) and xyloglucan (LM25) were present across the crops. The relative signals of the pectic polysaccharide-related MAbs were overall higher within the eudicotyledons. The relative epitope levels of HG (JIM7) and RG-I (LM6) were high within the hydroponate of tomato. The epitope levels of RG-I (LM6) were also high within the hydroponate of barley. An aliquot (9 mL) from each bucket was removed for one replicate, 1 mL 10x PBS was then added to each aliquot for the ELISA. The 1:25 dilution of the barley and maize hydroponates was used for the analysis; 1:5 dilution was used for pea, tomato and rapeseed. Data are a mean of three biological replicates. Twelve wheat plants were grown per bucket, per species, per replicate. A cut off point of 0.1 au was removed, any values below this level were regarded as having no signal (-), shading of heat map was across the species screened.

3.2.7 Polysaccharide epitopes released by the roots of crops remained present after concentrating

To evaluate the effects of concentrating the hydroponate of other species, the major epitopes were re-assayed after concentrating. Each bucket of hydroponate was filtered using Whatman paper filter to remove any particulates. The hydroponates of barley, maize, pea, tomato and rapeseed were concentrated from 9 L to ~200 mL using an ultrafiltration system that used a 30 KDa cut-off point membrane. After concentrating, the hydroponates were dialysed in dH₂O with six changes, and then freeze-dried. Hydroponates (10 µg/mL) were re-suspended in 1x PBS, and assayed using an ELISA. A similar profile of polysaccharide epitopes across the species was apparent when comparing the hydroponates to the concentrated hydroponates (Table 3.2 and Figure 3.5). Barley and maize concentrated hydroponates contained the highest relative epitopes of xyloglucan, xylan and extensin (Figure 3.5). The concentrated hydroponate of tomato contained the highest relative signal of AGP. The concentrated hydroponate of pea had the lowest epitope levels across the hydroponates screened. The concentrated hydroponates of barley and tomato had the highest relative epitopes of RG-I, whereas, for the other species the relative epitopes of RG-I were low (Figure 3.5).

Figure 3.5 I The major epitopes detected within the hydroponates of barley, maize, pea, tomato and rapeseed were retained after concentrating

Hydroponic medium was concentrated from 9 L to ~200 mL prior to ELISA. Quantities of total dried hydroponate that was above 30 KDa (10 µg/mL) were re-suspended in 1x PBS for assaying. Across the species screened, there was high variation within the released epitope profiles. The epitopes of xyloglucan were the highest in barley and maize. Barley had the most epitopes of xylan and extensin. RG-I (arabinan/AGP) related epitopes were high within barley and tomato hydroponates. Epitopes of AGP were highest in the hydroponate of tomato. Pea had the overall lowest MAb signals. In the hydroponate of rapeseed, the epitopes of xylan and xyloglucan dominated. Data shown are a mean of three biological replicates. The 1:25 dilution of barley and maize concentrated hydroponates, and the 1:5 dilution of pea, tomato and rapeseed concentrated hydroponates were used for the analysis. Error bars show standard deviation.

3.2.8 The epitope profiles of barley wild type and a root hairless mutant differed

Barley wild type (Hordeum vulgare cv. Pallas Andrew) and the bald root barley mutant (brb; Gahoonia et al. 2001), which lacks root hairs, were grown in perlite and vermiculite (50:50) for a week prior to hydroponics. After a week, barley were transferred to the hydroponics system, and grown for a further two weeks. The resulting hydroponates were concentrated, and assayed using ELISA. Twenty-nine antibodies raised against cell wall polysaccharides were used to probe the concentrated hydroponates. The only polysaccharide epitopes detected within the hydroponates were LM2 and MAC207, AGP-specific; LM1 and JIM20, extensin-specific; LM11 and LM28, xylan-specific and LM25-specific, xyloglucan. In general, the heat map demonstrated that the MAb signals were lower within the hydroponate of the barley root hairless mutant compared to the wild type (Table 3.3). The only MAb signal that was higher within the hydroponate of brb compared to the wild type was LM25, that recognises the epitopes of xyloglucan, was 4.7x higher (Table 3.3).

Table 3.3 I Heat map of the polysaccharides released from barley wild type (WT) and the bald root barley (brb) mutant as determined by ELISA

Twenty-nine monoclonal antibodies raised against cell wall polysaccharides were used to screen the hydroponates of three-week-old barley through ELISA. Hydroponate of barley was concentrated from 9 L to ~200 mL prior to ELISA. Overall, the signals from the MAbs are lower within the barley brb mutant compared to the wild type (cv. Pallas Andrew). The signal of LM25 is much higher within the concentrated hydroponates of the barley mutant. An aliquot (9 mL) from each bucket was removed for one replicate, 1 mL 10x PBS was then added to each aliquot for the ELISA. The 1:25 dilution of the respective concentrated hydroponates was used for the analysis. Data are a mean of three biological replicates. Twelve barley plants were grown per bucket, per species, per replicate. A cut off point of 0.1 au was removed, any values below this level were regarded as having no signal (-), shading of heat map was across the wild type and brb screened.

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3.3 Discussion

3.3.1 Monoclonal antibodies and hydroponic systems are useful tools to explore polysaccharides released by roots

For the first time, MAbs have been extensively used to directly probe the hydroponic medium of crop species. These highly sensitive molecular probes detected an abundance of AGP, extensin, xylan and xyloglucan within the hydroponates of the cereals screened, particularly from the roots of wheat. Within the hydroponic media of the eudicotyledons screened, there was more variation between the species for instance; there was an abundance of AGP within the hydroponate of tomato. However, within the hydroponate of pea the signals from the AGP-specific MAbs were low. The epitope profiles of the root body and concentrated hydroponate of wheat differed, suggesting that these polysaccharides released from roots were being produced to be secreted. Interestingly, the root growth of the wheat cultivars did not significantly differ, yet the release of polysaccharides from their roots did. This suggests that there is a possible genetic factor behind the secretion of these molecules. By growing plants hydroponically and concentrating their hydroponate (Table 3.1 and Figure 3.3), this investigation has developed a novel method of attaining sufficient material for detecting the presence of a wide range of polysaccharides released by roots. Screening the hydroponic medium of the crops used in this study has led to the understanding that each plant released a unique epitope profile (Table 3.1 and Table 3.2).

Prior to this study, time consuming methods had to be undertaken to identify the polysaccharides that were being released by roots. One commonly used method, involved excavating plant roots from the soil in order to collect root mucilage (Morel et al. 1986; Mounier et al. 2004). Another method involved collecting released polysaccharides from seedlings that were grown in the dark on moist filter paper (Read and Gregory 1997). After a set number of days, released polysaccharides could be collected by pipetting the droplets of mucilage that percolated through the root caps. Alternatively roots were suspended in sterile deionised water (Bacic et al. 1986; Moody et al. 1988; Osborn et al. 1999). These methods of collection would have placed plants under stress, which could inadvertently affect the rate and types of polysaccharides released by roots. After collection, monosaccharide composition and monosaccharide linkage analyses would have to occur to reveal which polysaccharides were present. Monosaccharide composition and monosaccharide linkage analyses are the standard physio-chemical methods employed to determine what monosaccharides were present and how they were linked together. MAbs are highly sensitive molecular probes that are able to detect minor components of which these physio-chemical methods may not detect (Lee et al. 2011). Additionally, immuno-based assays such as ELISA require less purification than physio-chemical methods (Lee et al. 2011). Thus these methods can be combined to develop a more detailed understanding of polysaccharides released by roots.

A major limitation of MAbs and CBMs is that the resulting signals must be carefully interpreted as the binding affinities of each molecular probe can vary (Pattathil et al. 2012; Gilbert et al. 2013). However, using commercial standards to quantify absorbance units generated by MAbs and CBMS could reduce this effect. Another limitation is that if a sample is abundant in molecules they can begin to compete the binding sites of MAbs and CBMs within an ELISA (Marcus et al. 2008). This molecular crowding can mask epitopes of polysaccharides giving a distorted understanding of what polysaccharide is present and in what amount. It has been reported that xyloglucan, xylan and mannan can be effectively masked by the present of pectin during fluorescence microscopy of the primary cell walls (Hervé et al. 2009; Marcus et al. 2010). Using higher dilutions of the sample can reduce the concentration of polysaccharides, and thus the masking of the epitopes. An additional factor to consider when using these probes is that there might be a molecule that is present within a sample that neither a MAb nor CBM recognises, which may lead to a narrow understanding of that sample. Thus careful interpretation is required when using these molecular probes.

Using a hydroponic system ensured that the crops were subjected to close to as natural conditions as possible, nevertheless, ensuring strict control conditions, and ease of isolating released polysaccharides. Hydroponics also ensures that only the roots are in contact with the medium. This makes certain that these polysaccharides are released from roots and not released from other sources, including the seed coat (Haughn and Western 2012). Despite the efficiency and ease of isolating polysaccharides, hydroponics still remains an unnatural system to grow plants. Nevertheless, studies have optimised hydroponic systems for growing many crops (du Toit and Labuschagne 2007; Giecornmelli et al. 2015). Organisations such as Thanet Earth are already successfully contributing to UK food production; frequently growing crops, including tomato, lettuce, cucumber and peppers, hydroponically that are more cost effective compared to field grown crops.

3.3.2 Polysaccharides released from the roots of wheat may be produced to be secreted into the rhizosphere

A diverse range of polysaccharides were released into the hydroponates of crops screened. Crops were selected because of their large root networks, maximising the probability of collecting sufficient material for biochemical analysis, but also as some of the species had already been identified to release polysaccharides (Bacic et al. 1986; Moody et al. 1988; Read and Gregory 1997). Crops were also selected due to their tolerance for hydroponic culture (Arnon 1983; du Toit and Labuschagne 2007), and for the potential applications of this research on their production. There was an abundance of xyloglucan, xylan, extensin and AGP with b-linked glucuronic acid in the hydroponate of wheat (Table 3.1). This confirms previous work that had detected the presence of AGP, xylan and xyloglucan through the use of monosaccharide linkage analysis (Moody et al. 1988). The glycoprotein extensin has not previously been detected within the exudates of plants. The role of extensin within exudate remains unclear, however, they are abundant within the primary cell walls of roots, vascular bundle, and at the surface of epidermal cells (Smallwood et al. 1995), making more prone to be sloughed off and released into the rhizosphere.

Reasons for the abundance of xyloglucan within the hydroponates of cereals remain unclear. When the hydroponates of three wheat cultivars were screened, there were differences in their epitope profiles (Table 3.1). This was also observed when growing two cultivars of barley (Table 3.2 and Table 3.3). These observations suggest that there is a genetic factor behind the release of these molecules, further alluding to their importance. Perhaps, this indicates these polysaccharides, particularly xyloglucan act as functional molecules outside of the root body. Another interesting observation is the difference in the epitope profiles of the root body and hydroponate of wheat (Figure 3.4). If these polysaccharides solely derived from lysed cell wall components then the epitope profiles of the polysaccharides would not differ within the root body, and the hydroponate. This suggests that wheat roots, in particular, are producing these polysaccharides to be released whether that is through constant release through active secretory mechanisms or border cells (Northcote and Pickett-Heaps 1966; Guinel and Gregory 1986; Stephenson and Hawes 1994; Mravec et al. 2017).

As root caps and tips release root mucilage to aid soil penetration, it may then be possible that the root hairs also release root mucilage to aid their penetration into soil as well as secure the rhizosheath. If root hairs release polysaccharides then this would greatly increase the surface area of which these molecules are secreted. This may explain the difference in epitope profiles of the root body and hydroponate of wheat. These single celled hairs have not been demonstrated to be involved in the secretion of any organic substances. However, there is a strong indication that root hairs play a role in releasing polysaccharides into the soil. From the barley wild type and root hairless mutant heat map (Table 3.3), the MAb signs were mostly lower within the mutant, suggesting they may be releasing polysaccharide. The only signal that was higher in the hydroponate of the mutant was from xyloglucan. Reasons for the higher signals of xyloglucan remain unclear, potentially highlighting the importance of xyloglucan as a released polysaccharide. Perhaps, polysaccharides released from roots are secreted by different regions of the root system for example, xyloglucan may be released by the root hairs, whereas, AGP and pectin are release by the root caps and tips. It would be interesting to examine the temporal dynamics of polysaccharides released from plant roots. Monitoring the levels of the major polysaccharide epitopes within the hydroponate of a plant, from seedling to senescence, would enable a glimpse into how their secretion alters over time.

Growing plants hydroponically does not reflect plants that are growing in soils. When plants are grown hydroponically their roots are not faced with high friction as they grow deeper into the medium. However, within soils the roots are subjected to high amounts of friction as they penetrate through the medium to get to the resources that they require. It would be interesting to determine how hydroponics affects the release of polysaccharides from roots and their composition. Since no other studies have used hydroponics to explore released polysaccharides, more research is required to understand these effects. These effects could be studied by growing wheat in an invert transparent soil such as glass beads, which could then be washed with KOH to extract the polysaccharides released from the roots.

Other polysaccharide epitope profiles were developed on two other cereal crops. These profiles indicate that the major polysaccharides within the hydroponate of barley and maize were of AGP, extensin, xylan and xyloglucan (Table 3.2). These polysaccharides (excluding extensin) released from maize roots have also been detected by other investigations, which use maize as a model for root mucilage (Bacic et al. 1986; Moody et al. 1988). This investigation determined that there was a lack of pectin within the hydroponates of the crops, with the exception of barley where RG-I was found. In general, a lack of pectin has also been determined within the root growth media of cereals (Table 1.1; Moody et al. 1988; Guinel et al. 2000; Sims et al. 2000), which conform to known grass cell wall biochemistry, where pectic polysaccharides are present in trace amounts (Nishitani and Nevins 1989; Carpita 1996; Vogel 2008). This supports the lack of pectin in the hydroponates of cereals tested. However, previous work has determined the presence of pectin within the root growth media of a number of eudicotyledons (Table 1.1, Ray et al. 1988; Osborn et al. 1999; Narasimhan et al. 2003). This investigation uncovered the presence of HG and RG-I within the hydroponate of tomato. However, the signals from the pectin-related MAbs were generally very low to not present across the other eudicotyledons. Additional research is required to further understand the differential in the detection of pectin uncovered in the hydroponates of eudicotyledons, and the detection of pectin within previous investigations. More research is also needed to understand the detected of RG-I (LM6; Table 3.2) within the hydroponate of barley.

There were no signals of LM8 (XGA) in the hydroponates probed. This is an interesting observation as LM8 has been shown to be specific to the root cap cells of pea and maize (Willats et al. 2004), which have also been used for this investigation. This may indicate that there were issues related to the solubility of XGA, which could be tightly attached to root cap cells. This may also be an indication that the root cap cells are not lysing within the hydroponic medium as roots are not subjected to friction caused by soil. This could support the hypothesis that these polysaccharides are being produced to be released. Further research is required to immuno-label the hydroponically grown roots using LM8 to determine if XGA is present within the root cap cells, as previously determined (Willats et al. 2004). If LM8 labels the root cap cells but is not detected within the hydroponate, this is a good indication that the roots are releasing polysaccharides not as a secondary effect but as a direct means. This would support their possible functional importance outside of the root body. If root mucilage, which contains AGP and pectin (Miki et al. 1980; Morel et al. 1987; Read and Gregory 1997), forms a highly viscous layer surrounding root caps and tips, it is conceivable that it remains at the root caps and tips of plants when grown hydroponically. Droplets of root mucilage were visible when plants were lifted from the hydroponic medium, as previously described (McCully and Sealey 1996; Read and Gregory 1997), but were disturbed when plants were harvested. Perhaps, some polysaccharides released, such as xyloglucan, are more soluble upon release compared to their more viscous counterparts, AGP and pectin. To confirm this hypothesis, plants could be grown on nitrocellulose sheets, which are highly absorbent, could be used to absorb polysaccharides released by roots. These nitrocellulose sheets could then be screened by MAbs to determine how far these molecules diffuse from the root body.

3.3.3 Eudicotyledons release different polysaccharide epitope profiles to their cell walls

The hydroponates of eudicotyledons typically contained relatively more pectic polysaccharides, corresponding with their cell wall biochemistry (Table 3.2; Carpita 1996; Vogel 2008). AGP, extensin and xyloglucan were also present in the hydroponate of eudicotyledons (Table 3.2). However, AGP was predominately detected by a different MAb in rapeseed (MAC207). There was also a high signal from MAC207 from the hydroponate of tomato (Table 3.2). One possible explanation for the different AGP-specific MAb signals is that the glycan proportion of the AGP detected is heterogeneous, which is well documented (Bacic et al. 1997; Kieliczewski 2001; Cosgrove 2005). Another study had also reported the presence of AGP, pectin and xyloglucan from pea root mucilage (Narasimhan et al. 2003), supporting the released epitope profile of pea developed by this study. The eudicotyledons appeared to have greater diversity of released polysaccharides within their hydroponates compared to the hydroponates of cereal. The hydroponates of the eudicotyledons contained polysaccharides that were similar to their cell wall components (Table 3.2; Moody et al. 1988; Vogel 2008). This was reinforced by previous research, which undertook monosaccharide linkage analysis on the released polysaccharides of cowpea (Moody et al. 1988; Read and Gregory 1997), Arabidopsis (Chaboud and Rougier 1984), cress (Moody et al. 1988; Ray et al. 1988; Osborn et al. 1999; Sims et al. 2010) Indian rhododendron (Moody et al. 1988) and lupin (McCully and Sealey 1996; Read and Gregory 1997).

3.4 Conclusion

Monoclonal antibodies are useful diagnostic tools for exploring polysaccharides released from plant roots into a hydroponic medium. For the first time, crops have been grown hydroponically to isolate their released polysaccharides. The major polysaccharides uncovered within the hydroponates of the crops screened were xyloglucan, xylan, extensin and AGP. Other polysaccharides were uncovered but were not represented across the species for instance; there was an abundance of RG-I within the hydroponates of barley and tomato. There was also a high detection of pectin (HG) within the hydroponate of tomato. There was a higher relative signal of xyloglucan within the hydroponates of cereal. On further analysis, the relative epitopes of AGP, extensin and xyloglucan were more abundant within the hydroponate of wheat compared to the root body. Additionally, there were was no detection of LM8 (XGA), which is specific to root cap cells, within the hydroponates screened. These results support the hypothesis that these molecules are being formed to be released by roots. When comparing the relative epitopes levels of barley wild type and the root hair-less mutant, xyloglucan was found to be higher within the hydroponate of the mutant. This suggests that the root hairs may play a role in the secretion of polysaccharide. The root growth of the three wheat cultivars grown did not significantly differ, yet the polysaccharides released into their hydroponates differed. This was also observed within the hydroponates of two cultivars of barley. These observations indicate that there may be a genetic factor involved in the release of polysaccharides by roots.

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