Chapter 6: General Discussion

6.1 Towards a new understanding of polysaccharides released by plant roots

From this investigation, it is hypothesised that polysaccharides released by roots have a complex architecture, which would reflects the cell walls where they had been released from. These polysaccharides could be released from continually lysed cell wall polysaccharides of root caps, root tips (Bacic et al. 1986; Read and Gregory 1997) and root hairs, as well as root border cells or root border-like cells (Driouich et al. 2013). These areas of release could all contribute to the amount and distribution of polysaccharide released by roots. When seedlings are establishing themselves in soil, they may exude more polysaccharide to develop a strong rhizosphere required for future growth. As a plant begins to mature they may release fewer of these molecules, as they have established their rhizospheres, only needing to maintain their rhizosheath when new roots grow into the soil.

Preliminary work had grown wheat for two-and-a-half months, and uncovered a gradual decrease in the detection of polysaccharides after three weeks (data not included). This is supported by previous research which uncovered that seedlings released more root mucilage compared to older plants (Chaboud 1983; Bacic et al. 1986; Moody et al. 1988; Read and Gregory 1997; Osborn et al. 1999). Rapidly developing the rhizosphere would increase the uptake of resources, particularly, at a time when a plant requires a large amount of resources for growth. Future work exploring the role of these polysaccharides should aim to compare the rate of which these polysaccharides are released from young and mature plants.

This investigation demonstrated that there was a higher signal of LM25 (xyloglucan) within the hydroponate of the barley root hair-less mutant (brb; Gahoonia et al. 2001). This initial screen has provided tantalising results (Table 3.1) that indicate root hairs may release polysaccharide. Immunofluorescence microscopy could be used to determine if these root hairs release polysaccharide in situ. For instance, Cadenza could be grown in sand; once they reached three-weeks-old the plant roots could be carefully excavated so that it preserved the rhizosheath prior to microscopy. MAbs LM1 (extensin), LM2 (AGP), LM11 (xylan) and LM25 (xyloglucan) could then be used to screen the sample of rhizosheath. Some research has suggested that some grasses are able to increase the thickness of their rhizosheath during periods of drought (Hartnett et al. 2012). The mechanisms underpinning their ability to regulate the thickness of their rhizosheath were undetermined. However, it is feasible to suggest that polysaccharides play a role in rhizosheath thickening. As root hairs are abundant on roots (Gilroy and Jones 2000) it may be possible that they aid with the thickening of the rhizosheath. Growing barley brb in sand and measuring the amount of particles adhered to their roots would be one way of exploring this hypothesis. Using such techniques as X-ray computerised tomography (CT; used at the University of Nottingham; Tracy et al. 2015; Daly et al. 2017) would enable accurate 3D imaging of the rhizosheath, and accurate quantification of the amount of soil adhering to roots.

Root mucilage may just be formed of AGP and pectin, which form viscous gels (Bacic et al. 1986; Moody et al. 1988; Read and Gregory 1997), whereas, other polysaccharides released by roots, as detected by this investigation, extensin, xylan and xyloglucan could be more diffuse in the soil. These more diffuse components could be involved in modifying the rhizosphere, enabling plants to extract more resources from soil. Modifications to the rhizosphere would include soil aggregation to secure the rhizosheath. While, the gel forming polysaccharides that produce root mucilage would solely lubricate roots as they penetrate through soil. The more soluble polysaccharides would be released across the entire root network, rather than the root caps and tips. To determine if the polysaccharides that are released by roots have different solubility, nitrocellulose sheets could be used to screen locations of secretion along the roots. Plants could be grown on nitrocellulose, which is highly absorbent for a few days. After growth, the nitrocellulose could be screened with MAbs to reveal where each polysaccharide is secreted. Attempts at growing Arabidopsis on nitrocellulose have demonstrated that pectin and AGP appear along the root surface, whereas xyloglucan and xylan appear more diffuse, moving away from the roots (data not included). However, developing nitrocellulose blots that had larger plants growing on including wheat was challenging. Issues included keeping roots in contact with the nitrocellulose, the moist nitrocellulose causing a run off effect where the polysaccharides covered the entire sheet, and determining an appropriate dilution to use for the MAbs which could quickly become overdeveloped.

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6.2 Categorising the putative multi-polysaccharides uncovered in this study

An issue of categorising the putative multi-polysaccharide complexes uncovered within this investigation arose. REC1 was initially uncovered from the hydroponate of wheat cv. Cadenza. Further immuno-chemical and physio-chemical methods were used to explore the biochemistry of this complex. When screening the hydroponates of two other cultivars of wheat (Avalon and Skyfall) a REC1-like molecule was detected. The term REC1-like was used as the immuno-based assays demonstrated that there were slight differences in the signal strengths of the MAbs between the cultivars. Whether REC1 released from Cadenza is the same as the other cultivars, more physio-chemical analysis through monosaccharide linkage analysis and possibly NMR is required. Moreover, REC1-like molecules were also uncovered within the hydroponates of barley and maize. Establishing whether REC1 released by wheat is the same (or not) as barley and maize is needed through physio-chemical methods, as outlined above. The exudates of more grass species needs to be screened to determine if releasing a REC1-like molecule is widespread across the grasses. Some evidence of REC1-like molecules was detected within the hydroponates of pea, tomato and rapeseed. More immuno-chemical and physio-chemical research is needed to determine if eudicotyledons release a REC1-like complex. Furthermore, a wider range of MAbs is needed to determine if eudicotyledons release a distinct multi-polysaccharide complex from grasses (REC1) and liverworts (REC2).

6.3 The possible structure of REC1

Results presented here have indicated to the presence of a multi-polysaccharide complex released by the roots of cereals. However, the structure of this complex remains to be fully understood. The monosaccharide linkage analysis undertaken by the Complex Carbohydrate Research Centre (CCRC) demonstrated that there was a high mixture of linkages and structural complexity within REC1. Clear signatures of AGP, extensin, xylan and xyloglucan were detected, confirming what the MAbs had been detecting. Previous investigations using wheat and maize grown in sterile conditions have also determined signatures from AGP, xylan and xyloglucan (Bacic et al. 1986; Moody et al. 1988). In addition to these signatures, past investigations had also determined signatures that do not have a clear polysaccharide signature, 6-linked Glcp and 2,4-linked Glcp (Moody et al. 1988). Other linkages detected within maize and cowpea, 3-linked Fucp, 2,3-linked Galp, 3,6-linked Galp and 2,3-Manp and 3,4-linked Galp (Moody et al. 1988) had also been described within this study that focused on wheat (Table 4.1). This investigation had determined novel signatures of arabinan, extensin, galactomannan, mannan, mixed linkage glucan, RG-I and RG-II. Considerably more work is necessary to understand how these linkages come together to form REC1 if indeed they are all part of a polysaccharide complex. Research using NMR-based techniques could be used to determine how they are linked. However, a larger amount of REC1 would need to be isolated from Cadenza, thus a more efficient method of isolating these polysaccharides is required. Once determining what polysaccharides are present, the next stage of work would need to explore how these polysaccharides were linked. For instance, is AGP linked to extensin to form a protein core that crosslinks with the other polysaccharide domains (Figure 6.1)? What kind of bonding links REC1, for example covalent linkage? Is REC1 in the form of a complex (Figure 6.1), or could it be a heterogeneous mixture of polysaccharides released by roots?

Within the linkage analysis there were unexpectedly high levels of glucose and mannose linkages. These linkages were previously detected in the fungi (Rhynchosporium secalis), which causes barley leaf scald (Pettolino et al. 2009). As the wheat was grown in non-sterile conditions, and that these linkages have not been described before in plants, it is possible that these linkages show indications of fungi cell wall components. Perhaps, these fungal components are from another fungi or bacteria of which linkage surveys of their cell walls have not been conducted. One exception to this is 2,3-linked Manp, which had been described in the medium of maize (Bacic et al. 1986). Therefore, another possibility is that these linkages are novel polysaccharides released by wheat roots. When discounting all previously described linkages in wheat, maize, cowpea, and barley leaf scald there were two linkages within this study (2,3,6-linked Galp and 2,4-Glcp), which had not previously been reported. Future work using the hydroponics system developed by this investigation would have to ensure that the hydroponate was sterile. Sealing the hydroponates within the buckets would reduce the risk of contamination by airborne fungi or bacteria. All equipment would also have to be autoclaved or chemically sterilised using bleach. Seeds would also have to be sterilised particularly, at the pre-hydroponics stage. A hydroponics system could also be grown within a sterile growth cabinet rather than a glasshouse. However, controlling humidity may be challenging.

A comparison of the linkages present in the root cell walls of wheat is required to fully understand the novelty of the polysaccharides released by roots. If a novel form of mannan is present within REC1 could an enzyme (endo-1,4-β-mannanase) be used to breakdown the backbone of this potential domain? If so what effect would this have on the structure of REC1? MAbs could be used to determine if there was an alteration in the four polysaccharide epitopes that form REC1, as determined by anion-exchange EDC. The consistent signal from LM2 throughout this investigation, which binds to β-glucuronic acid in AGP (Willats et al. 2004) has contrasted with the lack acidic residues uncovered during monosaccharide linkage analysis at the CCRC. Could REC1 contain a minor proportion of β-glucuronic acid which is below the level of detection of the monosaccharide linkage analysis? If so could an enzyme that targets this residue (β-glucuronidase) be used to degrade this residue to determine if it makes REC1 acidic during anion-exchange EDC?

Figure 6.1 I A schematic diagram of REC1

After anion-exchange EDC, sandwich-ELISA, monosaccharide composition analysis and monosaccharide linkage analysis it is proposed that REC1 is formed of an AGP core, which crosslinks with the other domains. The AGP core could have a close attachment of extensin to form a possible protein domain (dashed line). Polysaccharide domains of xylan and xyloglucan may be attached to the AGP core branching out. It remains to be determined if mannan is a domain of REC1 or whether it drives from fungal cell wall components. XG = xyloglucan.

It would be interesting to conduct surveys on Arabidopsis exudates. Using this model species would open the possibilities of exploring the roles, structures and biosynthesis of these polysaccharides released by roots through the use of mutants. Such mutants as xxt1/2 (encodes two xylosyltransferase genes) which lack the ability to produce xyloglucan (Cavalier et al. 2008), and XUT which lacks the acidic xyloglucan from root hairs (Peña et al. 2012a), would enable research to explore the effects of removing xyloglucan from the polysaccharides released by roots. Attempts had been undertaken to grow Arabidopsis hydroponically; however, these attempts were not successful. Additionally, using the model species Brachypodium distachyon (purple false brome) would provide results more related to grass (Draper et al. 2001). Within the crops screened in this study, the plants were young (about three-weeks-old). Research in the US and France have developed systems of growing Arabidopsis hydroponically, however, they only achieved this using plants close to maturity (A. Galloway, 2016. In person A. Driouich, 14 June). This research would require young plants (within three weeks) to maximise the collection of these polysaccharides for biochemical and physio-chemical analysis.

6.4 The possible roles for polysaccharides released by roots within the microbiome

Plant roots are a part of a complex ecosystem that contains a plethora of life. The microbiome of the rhizosphere has only begun to be described in detail. One such concept is the Wood Wide Web, which is a complex network of fungi hyphae and plant roots (Helgason et al. 1998). This interconnected network involves the shuffling of carbon to fungi in exchange for nutrients (phosphorous and nitrogen) and water, which is inaccessible to roots (Haichar et al. 2014; Rilling et al. 2015). These series of interactions have been shown to occur across a forest, where most of the plants are connected to this large network of fungal hyphae (Helgason et al. 1998; Haichar et al. 2014). It has even been demonstrated that these hypal networks can shuttle nutrients and carbon from plant to another, which are separated by tens of metres (Barea et al. 2002). It would be interesting to determine if polysaccharides released by plant roots have a role within this network. Could these polysaccharides be used as a source of energy for the microbiome of the soil?

Some research has suggested that polysaccharides released by plant roots can be accessed by the microbiome of soil (Mounier et al. 2004; Benizri et al. 2007; McNear 2013). Studies have demonstrated using the root mucilages of maize, pea, alfalfa (Medicago truncatula) and cowpea, as the sole carbon source, can be used to grow various species of rhziobacteria and arbuscular mycorrhizal fungi (May et al. 1993; Knee et al. 2001; Gunina and Kuzyakov 2015; Sun et al. 2015). One interesting observation of these microorganisms grown using root mucilage was that they increase the production of their exopolysaccharides (Gunina and Kuzyakov 2015; Sun et al. 2015). The numbers of bacterial cells also rapidly increased as a result of the increase in available energy (Knee et al. 2001; López-Gutiérrez et al. 2005). Plants that can undertake symbiosis with rhziobacteria such as pea and lupin, could release high amounts of glucomannan within their exudate to attract, and mediate the attachment of nitrogen-fixing bacteria to their roots (Hoflich et al. 1994; Knee et al. 2001). In response, rhziobacteria release various glucanases that breakdown parts of the cell wall of root hairs prior to symbiotic penetration (Peleg-Grossman 2007). These findings suggest that microorganisms can actively use these polymers as a carbon source. When isolated root mucilage from maize was added to agricultural soil, bacterial numbers rapidly increased followed by the numbers of bacterial species detected (Knee et al. 2001; Walker et al. 2003). Moreover, it has also been shown that there is a higher density of microorganisms within the rhizosphere (between 2% and 7% w/w) compared to the bulk soil (≤ 1% w/w; Dennis et al. 2010; Garcia-Salamanca et al. 2013). From the results of these studies, it is clear that polysaccharides from roots could be used by the microbiome of the soil as an energy source. However, it remains unclear whether this occurs in the rhizosphere. Future work should focus on addressing the following questions, what are the ecological consequences of plants releasing polysaccharides into soil? How will releasing polysaccharides be affected during climate change? Can plants regulate the amount and types of polysaccharide released into the rhizosphere?

It has been suggested that if infectious microorganisms evade border cells and defense molecules within the exudate, they could use polysaccharides released by roots as a source of energy (Cannsean et al. 2012). As an example, the plant pathogen Pythium blight (Pythium aphanidermatum) rapidly increased the uptake of polysaccharides from cucumber (Cucumis sativus) grown on agar in prior to infection (Zheng et al. 2000). When sufficient numbers of bacteria had developed infection occurred. Once infected, the rate of mucilage produced by the cucumbers was found to decrease, and is then re-directed to producing more Pythium (Zheng et al. 2000). However, there are still many unanswered questions between the interaction of infectious microorganisms and these released polysaccharides.

It has been speculated that different plant species release different levels and types of carbon into the rhizosphere (Morel et al. 1986; Ray et al. 1988; Sims et al. 2000). Furthermore, the diversity of bacteria was found to differ when the root mucilages of pea, alfalfa and maize were supplied to a mixture of bacteria (Mounier et al. 2004; Benizri et al. 2007). It may be possible that roots release a unique fingerprint of polysaccharide into the rhizosphere in order to modify the surrounding soil, to suit the particular needs of that species (Badri and Vivanco 2009; Miscallef et al. 2009). For instance, if a plant requires a particular interaction with a particular species of microbe, the plant could modify the amount, and type of polysaccharide it released. It is conceivable that these released polymers play a multitude of roles for the plant, from soil aggregation to developing symbiotic partnerships. It would be of interest to determine if polysaccharides released by roots could be detected by MAbs within a native (non-sterile) soil. Glyco-typing the rhizosphere could have important ecological impacts such as in forest restoration, where a soil could be tested to see which plants had been growing prior to a natural disaster, such as wild fire. Furthermore, an experimenter could glyco-type a soil to determine the presence of invasive species before it spreads. For this to occur a large database would have to be developed in order to accurately glyco-type the polysaccharides released by a particular plant. This glyco-typing could also be expanded to explore the microbial polysaccharides of rhizospheres. For example, do the microbiomes of the rhizospheres of plants differ?

One major problem faced by agriculture is the increasing amount of soil becoming eroded (Erktan et al. 2016). As the global population increases the amount of food produced must increase in order to feed the growing population. Back in the 1960s the Green Revolution occurred which increased the production of food by using a combination of new intensive agricultural practices, developing shorter stem plant cultivars that produce more grain, and the use of chemical fertilisers and pesticides (Evenson and Gollin 2003). In order to feed a larger global population a second green revolution is needed. Although the successes of the Green Revolution are clear, the intensive agricultural practises have led to a rapid increase in soil erosion (Evenson and Gollin 2003; Erktan et al. 2016), which results in more land being used for agriculture. By reducing soil erosion, less land is required for food production. Perhaps, soil erosion could be reduced by growing plants that have a high release of polysaccharides. Growing these plants may help to increase the abundance of soil aggregates, and thus reduce the risk of erosion or even recover soil that has been damaged. Moreover, the development of a soil conditioner based on commercial plant polysaccharides could, firstly reduce the soil lost to erosion, and secondly protect soil that is already in use and vulnerable to erosion. Developing a soil conditioner could also act as a natural fertiliser feeding the microbiome, in order to help grow crops without the need for artificial fertilisers such as phosphate rock, which is an unsustainable resource. Within 50 years’ phosphate rock is predicted to become depleted (Gilbert 2009).

Understanding how plants maintain and secure their rhizosheath may help future missions to the moon. By 2037 NASA aims to set up a Luna outpost, which must be self-sustaining, cultivating sufficient food for the inhabitants. Previous research which utilised moon regolith, comparable to glacial rock, collected from Apollos 11 to 15 uncovered that the Luna regolith could support plant growth when sufficient water and nutrients were added (Walkinshaw et al. 1970; Walkinshaw and Johnson 1971; Baur et al. 1974; Ferl and Paul 2010). No examinations were conducted on the aggregate size and stability of the regolith post-plant growth (A. Galloway, 2017. Email to J. Wheeler, 9 May). It would be interesting to add released polysaccharides and commercial based-polysaccharides to Luna regolith to explore possible effects on regolith aggregate potential, which was observed when using different rock types on Earth.

6.5 Released polysaccharides may have given early plants an evolutionary advantage

Before plants colonised the land, early soils would have consisted of weathered rock fragments (Huggett 1998). Once plants had started to colonise the land, the turnover of early plants would have greatly added carbon into the rock fragments, which would come to form the first soils (Arteaga-Vazquez 2015; Mitchell et al. 2016; Harholt et al. 2016). To extract resources including water and nutrients, which would have been readily accessible within the sea, plants had to develop an interface with this rocky substrate. Perhaps, early plants with a higher rate of cell wall lysis resulted in a stronger interaction between rhizoids and early soils. This early rhizosphere may have given early plants an advantage over others, enabling them to access resources from rock fragments.

Liverwort rhizoids have been shown for the first time to release polysaccharide, similar to their modern-day relatives. This indicates that releasing polysaccharide may be widespread across the plant kingdom. Liverworts were found not just to release individual polysaccharides but release a complex, formed of AGP-xyloglucan. Considerably more research, using monosaccharide composition and monosaccharide linkage analyses is required to fully explore the physio-chemical properties of the polysaccharides released by liverworts. However, the amount of polysaccharide collected from liverwort is limited by the slow growth and small size of the plant. A more efficient method used to grow liverwort and to isolate the polysaccharides from the medium is needed in order to further explore the biochemistry of the polysaccharides released by liverwort. Future work should also focus on how widespread releasing multi-polysaccharide complexes are. Do mosses, hornworts or ferns (Figure 4.18) release a polysaccharide complex similar to that of REC2 or REC1?

6.6 Limitations

There are a few limitations to the techniques that were used in this investigation that should be noted. The hydroponic system developed by this investigation was effective at isolating the polysaccharides released by roots. However, this system does not reflect a natural environment. Plant roots were submerged in a liquid medium unlike soil which contains a heterogeneous mixture of particles with varying amounts of air pockets. Another difference between hydroponics and soil is that roots are not subjected to continual friction as they penetrate into the soil. Additionally roots in hydroponics are under a constant stream of air bubbles which is required for aeration. As a result roots are slowly and constantly being agitated which does not happen when plants are grown in soil where they are static. If plants were grown in as close to as natural conditions whether in sterile soil or in a field this would mean that polysaccharides could not be isolated in sufficient amounts required for biochemical and physio-chemical analysis.

MAbs have been instrumental in the biochemical analysis of polysaccharides within the hydroponate of plants. Although MAbs are highly sensitive probes their antigen may be masked by other polysaccharides that are in larger amounts. Furthermore, the ELISA plate wells will only bind to a narrow selection of molecules within the hydroponate which is further compounded by the washing steps that were undertaken during ELISA. Together this means that there was a narrow focus on the hydroponate of plants. There may be additional factors unexplored that may influence the polysaccharides released. Moreover, MAbs cannot discriminate between their polysaccharide antigens with their oligosaccharide counterparts. All together the issues reported here have been considered and a compromise between the advantages and disadvantages of the techniques was developed for this investigation.

6.7 Conclusion

Results presented here have expanded our understanding of polysaccharides released by plant roots. Grasses were uncovered to release REC1, a putative multi-polysaccharide complex that may be formed of a protein domain, containing AGP and extensin, as well as domains of xylan, xyloglucan and possibly mannan. REC1 was able to promote the aggregation of soil, more so than commercially available polysaccharides, xylan and xyloglucan. A complex formed of AGP-xyloglucan (REC2) was also detected in the medium of liverwort. Despite this new appreciation of polysaccharides released by roots, much more work is needed to fully decipher the biochemical properties of these polysaccharides. Future research should focus on three key areas: the structure of REC1, how widespread releasing these multi-polysaccharide complexes are across the plant kingdom, and determine how these polysaccharides interact with the microbiome of the rhizosphere. There are still many unanswered questions that need to be address including, do root hairs release polysaccharide? How do plants regulate the rhizosheath? How is REC1 formed? What kind of bonds hold REC1 together? Can polysaccharides be detected within native soil? Growing plants with a high release of polysaccharide in land vulnerable to soil erosion could be a new method of preventing soil degradation. These plants may even regenerate soil that has succumbed to erosion. This soil conditioning, using in situ planting or commercial plant polysaccharides, may one day lead to sustainable food production on the moon, which NASA hopes to do by using the moon’s regolith.

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