Identifying genes that can increase downstream benefits from cereals

Identifying genes that can increase downstream benefits from cereals

Author: | Date: 09 Feb 2016

Background

Cereal grain production in Australia is essential to the Australian economy and is estimated to have a net worth of $9 billion for immediate producers (About the grains industry). However, this value could be greater as losses are incurred when growers need to consider issues such as pests and environmental stresses which inevitably affect final grain yield and quality, and consequently price. One effective strategy that scientists are investigated in attempt to relieve the damage caused by pests and environment is by studying the plant cell wall (PCW). PCWs encase all plant cells and are composed of a complex mesh-like network of different polysaccharides (sugars), protein, lignin. This is where most of the plant biomass is stored. PCWs have many functions for the plant and also in human applications:

  • Molecular level: cell-cell communication, nutrient transport, cellular growth
  • Plant level: plant growth, strength to support the plant, flexibility (resiting environmental forces such wind) and protection against environmental stresses as well as pathogen defence
  • Human uses: main source of carbohydrate, dietary fibre supplement, biofuel production, brewing and feed for livestock.
Xylans are the second most common PCW polysaccharide in the world, second only to cellulose. The xylan polysaccharides are characterised by a backbone of xylose sugars and various sugar decorations, these backbone decorations further subdivide the xylan group into smaller ‘sub-groups’, each subgroup has a specific type of backbone decoration. The structure of a polysaccharide (length, type of sugar comprising the backbone and decorating it) influence the final function of the polysaccharide, thus structure is related to function. The most well defined xylan structure is that which is found in wheat grain, arabinoxylan (Figure 1). Despite its abundance and importance, very little is known about how xylan is made. 

Figure 1: Schematic showing the structure of arabinoxylan found in wheat endosperm.

Figure 1: Schematic showing the structure of arabinoxylan found in wheat endosperm. 

Understanding how PCW polysaccharides are synthesised enables scientists to generate the exact polymer for the final end-use. By understanding how something is made, we are then able to use this knowledge as at tool to produce ideal polysaccharides for different end uses. For example, reducing the amount of polysaccharide (a type known as beta-glucan found in the starchy endosperm of barley grains) in brewing varieties will help to prevent the clogging up of filters, whilst increasing the same polysaccharide in human food varieties is important as it is a dietary fibre with many positive health benefits on regular consumption. One well known example of how studying PCW biosynthesis has translated into an end product is a variety produced by the CSIRO called BARLEYmax™. In the late 1990s scientists at CSIRO were able to use their knowledge of key genes involved in beta-glucan biosynthesis in combination with traditional breeding strategies to develop the high fibre wholegrain BARLEYmax™, which has twice as much fibre when compared to other cereal grains. The discovery of BARLEYmax™ has enabled grain growers to receive guaranteed prices for barley. It is also estimated to have saved the health system $17million per year by decreasing diseases that are alleviated by consumption of a high fibre diet. The beta-glucan story clearly demonstrates how the knowledge of key genes involved in its biosynthesis can be applied into the grain industry and consumers abroad.

An example of how xylan could be used to improve the outcome for cereal grain growers is evident when we look at what happens in the PCW when a plant is under pathogen invasion. Powdery mildew is a fungal infection of the leaf. Growers currently spray plants with fungicide in order to prevent infection but the pathogen is able to build up resistance which then diminishes our capacity to control the infectious disease. In 2012 The GRDC estimated the economic cost of powdery mildew infection (loss of crop and downgrading of malt barley to feed) to be more than $200/ha (GRDC factsheet, March 2012). With approximately 20 million hectares of grain sown each year, a loss of $200/ha is certainly a considerable amount. Resistant barley varieties have been identified and in 2014 Chowdhury et al., (2014) reported that a plant’s ability to deposit arabinoxylan at the site of infection is a crucial step in preventing fungal infection, and consequently plant resistance (Figure 2). Therefore, by understanding when and how xylan is made, scientists can influence particular characteristics for breeders to select, and for new varieties to be released.

Figure 2: Example of how plants use arabinoxylan to gain resistance against powdery mildew infection. When a fungus infects the leaf, the plants first line of defence is to form a papilla to block the fungus from penetrating. (A) In resistant lines, an effective papilla (EP) is deposited that contains a high amount of PCW polysaccharide, including arabinoxylan, cellulose and some callose. In susceptible varieties, an ineffective papilla (IP) is produced where insufficient amounts of PCW polysaccharide are deposited and therefore enabling the fungus to penetrate and infect. (B) Microscopy imaging showing the fungus (F) and the formation of an effective and ineffective papilla. Notice the difference in size of the effective and ineffective papillae. Adapted from Chowdhury et al., (2014).

Figure 2: Example of how plants use arabinoxylan to gain resistance against powdery mildew infection. When a fungus infects the leaf, the plants first line of defence is to form a papilla to block the fungus from penetrating. (A) In resistant lines, an effective papilla (EP) is deposited that contains a high amount of PCW polysaccharide, including arabinoxylan, cellulose and some callose. In susceptible varieties, an ineffective papilla (IP) is produced where insufficient amounts of PCW polysaccharide are deposited and therefore enabling the fungus to penetrate and infect. (B) Microscopy imaging showing the fungus (F) and the formation of an effective and ineffective papilla. Notice the difference in size of the effective and ineffective papillae. Adapted from Chowdhury et al., (2014).

Methodology

In an attempt to identify genes involved in xylan biosynthesis, this study proposes to use Plantago ovata, commonly known as psyllium or ispaghol. Although wheat is our major source of dietary arabinoxylan (a hemicellulose), the genome for wheat is yet to be published and the arabinoxylan present in its grain is difficult to extract. Barley also contains arabinoxylan but in smaller amounts. Our proposed model system produces a lot of xylan that is easy to access (stored in the seed coat and extruded, as mucilage, when the seed is placed in an aqueous environment (Figure 3A)), is easy to grow (maturing in 3-4 months) (Figure 3B), and has a relatively small genome making genetic studies easier. Interestingly, P. ovata has been used in many eastern medicines for centuries and is currently one of India’s largest exports. The husk is milled off to produce psyillium husk, which can then be used as a dietary fibre supplement or gelling agent in food manufacturing. Perhaps the most important factor in selection of P. ovata as the model species for studying xylan biosynthesis is that it produces a xylan that is similar to that which is found in wheat (Figure 4). Consequently, it may be possible that the genes identified in our model system will be transferrable to our cereal crop species.

To find candidate xylan biosynthesis genes, we are using a genetic mutagenesis approach (Figure 5). When we mutagenise the wild type seeds, this causes various deletions throughout the genome. Some of these deletions will be in genes involved in xylan biosynthesis and as a result xylan production will be obstructed. In order to find these genes we first need to identify those xylan-mutant individuals. P. ovata seed mucilage is predominantly composed of xylan. When using microscopic and chemical analyses we can observe any individuals with varying mucilage staining pattern or composition (compared to that of the wild type). If any changes are observed it can be implied that the xylan polysaccharide has been changed. Once mutants have been identified and characterised we then compare the genetics of the mutant plant to that of the wild type to identify which genes have been mutated and cause the observed phenotype. The genes can then be isolated to determine its function, then homologous genes that may be present in cereal crops such as wheat could be identified and investigated further.

Figure 3: (A) The seed coat of Plantago ovata extrudes a xylan-rich gel (mucilage) when it is placed into an aqueous environment, here it is stained pink for easy visualisation, scale bar = 1mm. (B) Plantago ovata grown in the glass house. The figure shows mature seed heads, which are ready to harvest 3-4 months after germination. The mature plant is approximately 40cm in height.

Figure 3: (A) The seed coat of Plantago ovata extrudes a xylan-rich gel (mucilage) when it is placed into an aqueous environment, here it is stained pink for easy visualisation, scale bar = 1mm. (B) Plantago ovata grown in the glass house. The figure shows mature seed heads, which are ready to harvest 3-4 months after germination. The mature plant is approximately 40cm in height.

Figure 4: Heteroxylan found in Plantago ovata seed mucilage. Despite P. ovata xylan appearing more complex in structure to that of wheat (Figure 1) the linkages that are present between the xylose and arabinose sugars are the same. This indicates that the enzymes, and consequently genes, responsible for synthesising the two polymers are equivalent and results found in the P. ovata system could then be transferred into our cereal crop species.

Figure 4: Heteroxylan found in Plantago ovata seed mucilage. Despite P. ovata xylan appearing more complex in structure to that of wheat (Figure 1) the linkages that are present between the xylose and arabinose sugars are the same. This indicates that the enzymes, and consequently genes, responsible for synthesising the two polymers are equivalent and results found in the P. ovata system could then be transferred into our cereal crop species.

Figure 5: Scehmatic outlining the general method used to generate and screen for P. ovata seed mucilage mutants. (A) Mutagenising the seeds causes random deletions into the plant’s genome. (B) When we plant out the mutagenised seeds, they will produce plants and consequently seed which are also carrying the mutations. (C) The seeds produced from the mutant plants can then be harvested and (D) screened to identify seed mucialge mutants, indicating the possiblity of mutations in xylan-assicoated genes.Two mutants have been identified and show very different phenotypes: hikikomori shows a very compact mucilage strucutre whilst the mucilage extruded by the fugitive mutant is much more dispersed. (D) Scale = 1mm.

Figure 5: Scehmatic outlining the general method used to generate and screen for P. ovata seed mucilage mutants. (A) Mutagenising the seeds causes random deletions into the plant’s genome. (B) When we plant out the mutagenised seeds, they will produce plants and consequently seed which are also carrying the mutations. (C) The seeds produced from the mutant plants can then be harvested and (D) screened to identify seed mucialge mutants, indicating the possiblity of mutations in xylan-assicoated genes.Two mutants have been identified and show very different phenotypes: hikikomori shows a very compact mucilage strucutre whilst the mucilage extruded by the fugitive mutant is much more dispersed. (D) Scale = 1mm.

Results and discussion

We have successfully generated a mutant population and identified two fundamentally different mucilage mutants, hikikomori and fugitive (Figure 5D). That is, the genetic mutation appears to affect different points in the xylan biosynthetic pathway. It is of great interest that the two mutants have different characteristics as this enables us to investigate more than one point in the pipeline of xylan biosynthesis, which is a multi-step process.

The mutant, hikikomori (Figure 5D), shows a very compact mucilage phenotype. Chemical analysis on the sugar content of the extruded mucilage shows that the composition appears to resemble that of the wild type (Figure 6). Interestingly, further microscopic analysis using a probe that binds specifically to arabinoxylan reveals the abundance of this polysaccharide in the wild type mucilage (Figure 7A), but when the same probe is used in hikikomori a lack of labelling is evident (Figure 7B). This indicates to us that the probe is unable to bind to the xylan that is present in hikikomori. Perhaps due to the compact nature of the extruded mucilage, the probe is unable to penetrate into the mucilage. This finding suggests that the genes that have been mutated in hikikomori are associated with the final steps in polysaccharide biosynthesis: assembly of the sugar units (arabinose and xylose) into a longer more complex polymer have been affected.

Chemical and microscopic analysis of seed mucilage obtained from mutant fugitive shows astounding differences to that of both wild type and hikikomori. Compositional sugar analysis shows a considerable reduction in the amount of xylose and arabinose sugars (Figure 6); the sugars that when joined together make up the arabinoxylan polysaccharide. This supports the appearance of fugitive’s ‘dispersed’ and ‘soluble/dissolvable’ mucilage phenotype because a lack of arabinoxylan, which comprises 90 per cent of the mucilage, has meant that the mucilage has lost its structural properties. Contrasting to hikikomori, it appears that the mutation that is causing the fugitive phenotype has an affect very early in the xylan biosynthetic pathway as very little arabinose and xylose are detected, indicating that the plant is unable to produce these sugar precursors.

Material for genetic analyses has already been successfully obtained and sequenced. We are now analysing this data to find the causal mutations. Preliminary results suggest some key PCW synthetic genes may be implicated, this data is being further investigated. We have also been able to collect sample from different stages of development. These different stages will give insight into the timing of key genes: in the example of arabinoxylan being deposited at the site of powdery mildew infection, the data gathered from different developmental stages could be used to predict which genes are required at what stage therefore providing further information to aid in the breeding of resistant varieties.

Figure 6: Sugar analysis of the extruded seed mucilage reveals that mutant, hikikomori, has a similar sugar composition to that of the wild type. The other mutant identified, fugitive, has lower amounts of xylose (Xyl) and arabinose (Ara). These two sugars are what makes up arabinoxylan.

Figure 6: Sugar analysis of the extruded seed mucilage reveals that mutant, hikikomori, has a similar sugar composition to that of the wild type. The other mutant identified, fugitive, has lower amounts of xylose (Xyl) and arabinose (Ara). These two sugars are what makes up arabinoxylan.

Figure 7: Arabinoxylan labelling (observed as the bubble-like structures protruding from the seed) reveals that mutant, hikikomori, has a different structure of arabinoxylan. We know that is is present due to sugar analysis (Figure 5) but when we use the same binding molecule in wild type and hikikomori, it does not label hikikomori indicating that although the arabinoxylan sugar units are present, they may be assembled in a different structure. Scale bar = 0.5mm.

Figure 7: Arabinoxylan labelling (observed as the bubble-like structures protruding from the seed) reveals that mutant, hikikomori, has a different structure of arabinoxylan. We know that is is present due to sugar analysis (Figure 5) but when we use the same binding molecule in wild type and hikikomori, it does not label hikikomori indicating that although the arabinoxylan sugar units are present, they may be assembled in a different structure. Scale bar = 0.5mm.

Conclusion

Xylan is one the most prominent PCW polysaccharides in the world. The study of how this vital PCW polysaccharide is synthesised is sure to generate influential down-stream affects in many different industries. This study aims to identify genes involved in xylan biosynthesis using a mutagenic genetics approach and P. ovata. Two very interesting mutants have been identified and characterisation of the xylan-rich seed mucilage shows that the two mutants show differences in composition and structure. Our research has already made important findings in regards to the mutants and we are currently interrogating the genetic data to identify causal genes. 

To better iterate how this study can be applied, I would like to bring your attention back to the BARLEYmax™ and powdery mildew examples, but this time proposing a hypothetical future application for cereal crop varieties with different arabinoxylan content. Genes identified that are causal of the hikikomori mutant phenotype (compact mucilage) could be used to breed plants that produce a ‘stronger’ papilla and therefore be resistant to powdery mildew infection. Genes causal of the fugitive phenotype (dispersed) can be used in the dietary fibre supplement industry. Psyllium husk is currently used as a bulking agent (an insoluble dietary fibre), by incorporating a mixture of this ‘traditional’ psyllium husk with a more soluble psyllium husk. We would be able to see systemic benefits in reducing type II diabetes, cardiovascular diseases, colon cancer and high blood pressure which are effects of consuming soluble fibres. The increase in ‘solubility genes’ can also be used to develop brewing varieties whose grain endosperm content does not clog up filters. The study of beta-glucan has aided in the development of BARLEYmax™, estimated to be worth $305 million per year due to costs saved in healthcare. As arabinoxylan is the second most prominent PCW polysaccharide its unravelling story will no doubt, at the very least, be as intriguing as that of BARLEYmax™ to everyone involved in the cereal grain production line from breeders, growers, industry and end consumers.

Useful resources





Arabinoxylan improves barley resistance to powdery mildew infection: Chowdhury et al., (2014) Differential accumulation of callose, arabinoxylan and cellulose in nonpenetrated versus penetrated papillae on leaves of barley infected with Blumeria graminis f. sp. hordei. New Phytologist 204: 650-660

Plantago ovata: Dhar et al., (2005) Plantago ovata: genetic diversity, cultivation, utilisation and chemistry. Plant Genetic Resources 3: 252-263

Xylan: Ebringerova and Heinze (2000) Xylan and xylan derivatives – biopolymers with valuable properties. Macromolecules Rapid Communication 21: 542-556

Acknowledgement

Funding for this work was provided through the GRDC Project URS10490 and their support gratefully acknowledged.

Contact details 

Jana L. Phan
ARC CoE in Plant Cell Walls
University of Adelaide
Waite Campus, Urrbrae S.A. 5064
08 8313 6501

GRDC Project Code: URS10490,