Hetero-trans-b-glucanase (HTG), a unique cell-wall remodelling enzyme from Equisetum: action and potential to enhance mechanical properties of cereals

Lead Research Organisation: University of Edinburgh
Department Name: Sch of Biological Sciences

Abstract

BACKGROUND AND PURPOSE
We recently discovered a unique enzyme (HTG or hetero-trans-b-glucanase), found only in a group of non-flowering plants, the horsetails. Flowering plants lack HTG even though their cell walls contain the chain-like molecules which, at least in the test-tube, HTG can cut and re-join. We now aim to discover (a) what good HTG does horsetail plants, (b) the full range of 'cutting and re-joining' reactions that HTG can achieve, (c) what happens when HTG from horsetails is artificially transferred to crop plants. We predict that the horsetail enzyme will endow flowering crops, e.g. wheat, with the ability to strengthen their stems in a manner hitherto only available to horsetails. Such crops may acquire improved resistance to lodging (storm damage).

OBJECTIVES AND EXPECTED OUTCOMES
Remarkably, horsetail HTG is the only known enzyme from any living thing that can 'cut and re-join' molecules of cellulose, the major constituent of plant cell walls. It can graft a cellulose chain onto a chain of a different cell-wall building material called xyloglucan. HTG can also graft chains of a third such material (MLG or mixed-linkage glucan) onto xyloglucan. HTG can thus create cellulose-to-xyloglucan and MLG-to-xyloglucan linkages. The resulting 'hybrid' polymers are thought to strengthen horsetails. We will discover exactly when and where HTG is produced, and such linkages are formed, in horsetails. This will potentially give clues about HTG's natural roles. We will also discover what new reactions HTG can catalyse when mixed in the test-tube with diverse plant cell-wall polysaccharides. This may afford new 'hybrid' polymers, which when scaled up may be commercially valuable new materials. To further our fundamental knowledge of HTG, we will also investigate which of the enzyme's amino acids are important for its ability (in the test-tube) to re-configure cellulose and MLG.
A major part of this project involves artificially introducing the horsetail's HTG activities into flowering plants, including both dicotyledons and cereals, and measuring the consequences. Our industrial collaborators (Bayer CropScience) will do this work in the case of wheat. We predict that any crop plants genetically transformed in this way will be able to create cellulose-to-xyloglucan linkages in their cell walls, and that cereals (which, unlike dicots, possess MLG as well as cellulose and xyloglucan) will in addition be able to make MLG-to-xyloglucan linkages. We will test these predictions experimentally. We will also test whether the HTG-endowed flowering plants are stronger, and whether they have an altered shape or size. We will quantify the plants' mechanical strength by measuring the force required to bend or break their stems. Any changes to the molecular architecture a plant's cell walls are likely to affect its growth and strength because of the pivotal roles that cell walls play in dictating these features.

BENEFICIARIES OF THE PROJECT
Cereal varieties with stronger stems often suffer less lodging, but such strengthening is usually achieved by the plant growing thicker stems at the expense of lower grain yield. Artificially giving cereals HTG may form novel inter-polymer linkages in the cell wall and confer similar strengthening without significant increases in stem biomass and thus without compromising the harvest. Modifying cereals in this way would benefit plant breeders and farmers, as well as the general public, by improving the reliability of grain production in a changing climate as storms and heavy rains become more frequent. In addition, increasing knowledge of HTG's ability to reconfigure biomass materials, especially cellulose (the world's most abundant organic substance), offers biotechnologists novel opportunities to create new materials (e.g. for specialist papers and medical applications) via non-polluting 'green' processes.

Technical Summary

Plant cell walls contain transglycanase activities that 'cut and paste' xyloglucan (XyG), xylan, mannan and mixed-linkage glucan (MLG) chains. Classic XyG-acting transglycanases (XTHs) potentially contribute to wall architecture. Until recently, no cellulose-acting transglycanase was known. We discovered and sequenced HTG, an Equisetum enzyme that preferentially catalyses hetero-transglycosylation with cellulose or MLG as donor substrate and XyG as acceptor (m/s submitted). HTG's ability to make cellulose-XyG and MLG-XyG bonds may be valuable, both for functionalising biomass post harvest and for strengthening living crops. We will explore HTG's natural role in Equisetum, further define its in-vitro and in-vivo catalytic repertoire, and test the effects of an HTG transgene on the growth, morphogenesis and mechanical properties of Arabidopsis, wheat and maize. Wild-type angiosperms lack HTG but possess its substrates (cellulose + XyG in dicots; these + MLG in cereals), so we predict that transgenic HTG would act in crops. We will test this prediction. Effects of heterologous HTG may also add to our fundamental understanding of wall architecture. By site-directed mutagenesis, we will test the contribution of what are predicted (by 3D modelling) to be the 3 key amino-acid substitutions that 'converted' an Equisetum XTH into HTG. The results may suggest whether it is feasible to confer 'HTG' on crop plants through directed mutation of endogenous XTHs. In collaboration with Bayer CropScience we will test whether HTG expression in crops minimises lodging by strengthening cell walls via cellulose-XyG or MLG-XyG linkages previously confined to Equisetum. The project will exploit our unique expertise and recent discovery of HTG to perform proof-of-concept studies into crop improvement and to explore HTG's potential for post-harvest synthesis of novel bio-materials (e.g. speciality papers) via environmentally friendly biotechnological (synthetic biology) approaches.

Planned Impact

Most of the proposed work is specifically aimed at generating applicable outputs with (A) agricultural and (B) industrial impact - the former directly during this project, the latter for future exploitation.
(A) Agricultural impact. Potential beneficiaries of our in-planta efforts to enhance lodging resistance in angiosperms, by introducing the Equisetum HTG gene, are seed companies (including, but not limited to, Bayer CropScience), farmers and foresters, and ultimately the public through improved food security. Via our licence agreement, there is an engagement from Bayer to explore the potential impact of the technology - most immediately in the wheat seed market. Extension of the technology to other commercial plants (cereals, gymnosperms and dicots, including cotton fibres) will be of interest to Bayer and other plant breeding companies. Translating the knowledge gained into strategies accepted by the general public is realistic since we believe that endogenous angiosperm/gymnosperm XTH genes can be converted to 'HTG' genes via directed mutation without any need of genetic transformation. This seems feasible since it appears (and we will check in the present project) that converting an XTH to an HTG requires at most 3 amino-acid substitutions.
Our proposed work on HTG is relevant to BBSRC's Strategic Priority of living with environmental change (increasing frequency of heavy rain/wind damage, and thus lodging). The work is also relevant to companies involved in timber and bioenergy, since the results may be equally applicable to trees (including conifers) and biomass grasses (e.g. Miscanthus).
(B) Bio-materials/biomass impact. Our project does not specifically target the functionalisation of polysaccharides post-harvest; however, companies involved in this arena will find new potential in our work for synthesising novel bio-materials via 'green' synthetic biology. HTG creates novel 'hybrid' polysaccharides e.g. cellulose-XyG or MLG-XyG, which may have commercial potential for functionalising cellulose (the world's most abundant organic resource) and other plant biomass constituents. Characterising HTG's catalytic repertoire in vitro will offer scope for enzyme companies (e.g. Novozymes and Unilever) to market HTG. Knowledge gained in the project will also suggest to biomass users (e.g. CelluComp) ways to create bio-materials with new mechanical properties. HTG's ability to manipulate cellulose also has low-volume high-value potential, e.g. to manufacturers of speciality papers. For instance, HTG could be used to make papers with XyG-linked cargoes covalently bonded within the paper's cellulose fibres. Of numerous possible applications, speciality papers with indelible, xyloglucan-based 'ink' for use in legal documents or bank-notes would be good examples. These considerations augur well for finding industrial collaborators to take up our 'post-harvest' ideas.
Recent examples illustrate that we successfully concluded such deals. We work closely with ERI to disseminate/market information, with publicity materials summarising the main outcomes in an accessible manner. Through such marketing activity, some of the plant enzymes discovered in our BBSRC 'SCIBS' project are currently being explored for potential use in collaboration with a company who have a need for enzymes not found in mammalian tissues.
Other beneficiaries include basic scientists: new information on inter-polysaccharide bonding and its effects on wall mechanics will further our understanding of plant cell-wall architecture.
A clear beneficiary of this project will be the UK science-base, through the training of a chemically-minded postdoctoral plant biologist.
Samples of the novel substrates (including radiochemicals) generated during this project will be made available to the cell-wall and biomass communities e.g. for research use.

Publications

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Herburger K (2020) Defining natural factors that stimulate and inhibit cellulose:xyloglucan hetero-transglucosylation. in The Plant journal : for cell and molecular biology

 
Description The work is aimed at understanding and exploiting EfHTG (hetero-trans-b-glucanase), an enzyme thought to be unique to the 'primitive' land-plant genus Equisetum which we predict should be capable of catalysing useful reactions if expressed in commercial crops.
To allow us to track the distribution of HTG both in Equisetum plants and in transgenic angiosperms, we have sourced and bought a suitable ultra-violet fluorescence microscope, and designed two EfHTG-related epitopes and taken delivery of two antibody preparations. These antibodies will enable us to map the HTG protein in botanical specimens.
We have transformed two flowering plants (wheat and Arabidopsis) with the Equisetum gene, EfHTG, and verified its expression at the mRNA level.
We have tested extracts of the transgenic wheat and Arabidopsis plants, and untransformed controls, and shown that the former possess all three enzyme activities [cellulose:xyloglucan endotransglucosylase (CXE), mixed-linkage glucan:xyloglucan endotransglucosylase (MXE) and xyloglucan:xyloglucan endotransglucosylase (XET)] associated with the Equisetum protein, EfHTG.
We have developed a new assay for CXE action in living Equisetum tissue, and shown that this enzyme does indeed act on native cellulose to a limited extent, especially in dormant tissues.
In our studies aimed at further characterising EfHTG in vitro, we have produced two preparations of EfHTG: His6-tagged EfHTG in the yeast Pichia pastoris and GSH-tagged EfHTG in the bacterium E. coli. These two preparations are expected to be glycosylated and non-glycosylated, respectively, and we can now therefore test the contribution of sugar groups to the EfHTG's enzyme activities.
In our studies aimed at further characterising EfHTG in vitro we have also documented effects of competing oligosaccharides on XET, MXE and CXE activities of Pichia-expressed EfHTG and a related EfXTH (the latter is an Equisetum enzyme that exhibits very predominantly XET rather than MXE and CXE activity). We find remarkable differences between these two Equisetum-encoded proteins in their susceptibility to inhibition by specific oligosaccharides.
To probe the roles of specific amino acid residues in the EfHTG protein, we have achieved site-directed mutagenesis of HTG in E. coli, and are currently working to define the XET, MXE and CXE activities attributable to each targeted residue. This will allow us to explain the structural features that confer on EfHTG its unique MXE and CXE activities which led us to explore this new enzyme.
Exploitation Route It is hoped that angiosperm crops transformed to express the Equisetum enzyme, EfHTG, may have agronomically valuable traits, especially in connection with resistance to mechanical damage by wind and rain.
In addition, it is hoped that the enzyme itself can be used industrially to manipulate and improve the properties of cellulose-related polysaccharides. This aspect is currently [Feb-July 2020] being explored with IAA (Impact Acceleration Account) funding.
Sectors Agriculture, Food and Drink,Chemicals,Energy,Manufacturing, including Industrial Biotechology

 
Description The work has enabled BASF (formerly Bayer) to test the effect of inserting the HTG gene into a cereal crop on the growth, development and strength of the crop and they are currently exploring the commercial value of this alteration. In addition, several companies are in discussions with us on exploiting HTG as an agent to improve the quality of cellulose-based materials, especially paper/pulp and cotton fabrics. This is currently [Feb-July 2020] under investigation on a 6-month IAA (Impact Acceleration Account) project with the active support of timber, pulp, fabric and detergent companies.
First Year Of Impact 2019
Sector Agriculture, Food and Drink,Manufacturing, including Industrial Biotechology
Impact Types Economic

 
Title Biomechanical properties of plant materials. 
Description Method for assaying the biomechanical properties (strength, toughness...) of plant stems. 
Type Of Material Technology assay or reagent 
Year Produced 2017 
Provided To Others? No  
Impact Manuscript in preparation. 
 
Title Method for detection of cellulose:xyloglucan endotransglucosylase (CXE) action in living plant tissues. 
Description Method for detection of cellulose:xyloglucan endotransglucosylase (CXE) action in living plant tissues. Discrimination between in-vivo CXE, MXE and XET actions. 
Type Of Material Technology assay or reagent 
Year Produced 2017 
Provided To Others? No  
Impact First discovery of CXE action in vivo -- i.e. action of an endogenous Equisetum enzyme to catalyse the "cutting and pasting" of native cellulose chains in vivo. First evidence for an enzyme that modifies cellulose in vivo other than hydrolysis. Manuscript in preparation. 
 
Description Cellulose aerogel 
Organisation Mines ParisTech
Country France 
Sector Academic/University 
PI Contribution Assayed the ability of cellulosic aerogels to serve as donor substrate for CXE action of HTG
Collaborator Contribution Supplied the aerogel
Impact Manuscript in preparation. Interdisciplinary: carbohydrate chemistry, enzymology
Start Year 2017
 
Description Tim Stratford 
Organisation University of Edinburgh
Country United Kingdom 
Sector Academic/University 
PI Contribution Supply of transgenic plant material
Collaborator Contribution Design and execution of biomechanical measurements
Impact Measurements of biomechanical properties of transgenic angiosperm plant material.
Start Year 2018
 
Title Antibodies against Equisetum HTG. 
Description Antibodies against Equisetum HTG. Valuable for locating the distribution of HTG in plant material. 
Type Of Technology New Material/Compound 
Year Produced 2016 
Impact Localising the distribution of native HTG in Equisetum and of heterologously expressed HTG in wheat and arabidopsis. 
 
Title Novel sulphorhodamine-labelled xyloglucan oligosaccharides. 
Description Novel sulphorhodamine-labelled xyloglucan oligosaccharides. Valuable model substrates for exploring the cytological distribution of CXE, MXE and XET activities in plant material. 
Type Of Technology New Material/Compound 
Year Produced 2016 
Impact Used this fluorescent substrate to cytologically localise enzyme action (CXE, MXE, XET) in living plant material. 
 
Title Novel tritium-labelled xyloglucan oligosaccharides, especially those of degree of polymerisation >14. 
Description Novel tritium-labelled xyloglucan oligosaccharides, especially those of degree of polymerisation >14. Valuable model substrates to explore the acceptor-substrate specificity of HTG and other XTH-like enzymes. 
Type Of Technology New Material/Compound 
Year Produced 2017 
Impact Discovered the acceptor substrate specificity of HTG, showing that it prefers larger oligosaccharides which more closely resemble the xyloglucan polysaccharides of the plant cell wall. 
 
Title Transgenic angiosperm plants heterologously expressing Equisetum HTG 
Description "Material" = plant material. Transgenic arabidopsis plants heterologously expressing Equisetum HTG, including arabidopsis that is overexpressing xyloglucan biosynthesis genes. Transgenic wheat plants heterologously expressing Equisetum HTG, including wheat that is overexpressing xyloglucan biosynthesis genes. 
Type Of Technology New Material/Compound 
Year Produced 2017 
Impact Tested effect of Equisetum HTG expression on biophysical and biochemical properties of angiosperm plants.