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

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.

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.

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.