Xyloglucans xyloglucan endotransglucosylase (XET) activity and arabinogalactan-protein (AGP)-like molecules: a new inter-relationship

Each cell in a plant has an outer cell wall, which may be either stretchable (allowing the cell to grow, e.g. in a young beanstalk) or not (e.g. in an old leaf). Our experiments aim to discover how the plant can change its cell walls (making them more, or less, stretchable) and thus how the plant is able to increase and decrease cell growth. This ability is important so that, for instance, a seedling developing in bright light can produce short stocky stems and large leaves, efficient for photosynthesis, whereas one developing in the dark can grow a long thin stem (with a better chance of reaching any light) but only small leaves. Growing big leaves in the dark would be wasteful because they wouldn't be able to photosynthesise. Cell walls also glue neighbouring cells together, and so changes in the walls can dictate whether or not a plant tissue softens, e.g. in ripening fruit. Walls of young plant cells (those potentially able to grow) are built of several kinds of string-like structural molecules called polysaccharides. Two important ones are cellulose and xyloglucan. Our lab has been at the forefront of discovering, and understanding the role of, enzymes called xyloglucan endotransglucosylases (XETs). These catalyse a reaction in which the string-like xyloglucan molecules are cut and then re-joined. The cutting/re-joining process may be necessary (1) for new xyloglucan molecules to become bonded into the wall while it is being built, and (2) for an existing cell wall to loosen its structure (enabling cell growth). Work in other labs had highlighted another interesting class of polymers present in and around the plant cell wall. These are the arabinogalactan-proteins (AGPs), complex molecules made of carbohydrate and protein. They can bind to certain other carbohydrates, but any benefit of this binding so far as the plant is concerned is unknown. AGPs make up only a small proportion of the weight of the cell wall. They are not true structural wall components and are not enzymes; however, various experiments show that they somehow increase the ability of the wall to stretch and are thus important in enabling cell growth. We do not know how AGPs affect wall stretchability. However, our HYPOTHESIS is that AGPs act by increasing the rate at which XETs catalyse their cutting/re-joining reaction. We recently showed that some AGPs and related molecules can indeed promote XET action in the test tube. We will try to strengthen and extend the evidence for this hypothesis. Starting with cauliflowers and related plant tissues, we will purify and work out more details of the chemical structures of AGPs. We will also purify several different XETs from these tissues. We will then measure how fast each XET can catalyse its cutting/re-joining reaction in the presence of each different AGP. We will further test the new hypothesis by seeing whether Yariv antigens (chemicals that inactivate AGPs) stop XETs working in living plant tissues. We will also test whether, and how, AGP molecules bind to XETs and/or to xyloglucans. The knowledge gained would open the way to identifying genes that regulate the plant cell wall's properties, and thus in the future could give us the ability to control genetically or biotechnologically the growth and development of crops.

We aim to provide basic new information on the mechanisms by which plant cells regulate the assembly and loosening of their primary walls. Xyloglucan is a major hemicellulose of primary cell walls that hydrogen-bonds to, and probably tethers, cellulosic microfibrils. XTHs (xyloglucan endotransglucosylase/endohydrolases) are wall-localised proteins with two distinct enzymic activities: XET and XEH, catalysing the inter-polymeric endotransglycosylation of xyloglucan and the endo-hydrolysis of xyloglucan respectively. XET activity is thought to contribute to the integration of newly-synthesised xyloglucan chains into the inner face of the wall and also to the re-structuring of existing wall-bound xyloglucan chains. The latter process in particular may contribute to wall loosening and hence to cell expansion and tissue softening. Arabinogalactan-proteins (AGPs) are high-carbohydrate glycoproteins (i.e., proteoglycans; often 85-95 per cent sugar residues, usually rich in Gal, Ara and GlcA) with a short polypeptide backbone usually rich in Hyp, Ala, Gly, Ser and Thr, and often associated with a phosphorylated GPI anchor. AGPs occur mainly at the plasma membrane/wall interface, in the wall itself and in extracellular secretions. AGPs have been strongly implicated as promoters of wall loosening and hence cell expansion. The inner face of the wall, adjacent to the plasma membrane, is a particularly significant location because it is probably the load-bearing stratum of the wall. However, no widely accepted mechanism has yet been proposed to explain biochemically how AGPs influence irreversible wall extensibility. We now wish to study the interactions between XTHs and AGPs. Takeda & Fry (2004 and unpublished data) showed that AGPs and partially characterised AGP-like molecules from cauliflower florets can promote the XET activity of de-salted XTHs. Our central HYPOTHESIS is therefore that AGPs interact with XTHs and promote their XET activity, thereby regulating, spatially and temporally, wall-loosening and/or assembly. We will purify and further characterise several specific XTHs and AGP-like molecules from various crucifer tissues (Arabidopsis cell cultures, cauliflower florets and etiolated Brassica hypocotyls), and quantitatively assay the effect of various AGPs on various XTHs. We will further explore the central hypothesis by testing the effect of beta-Glc3-Yariv reagent (which binds to and inactivates many AGPs), in comparison with that of alpha-Gal3-Yariv reagent (which does not), on the in-vivo action of XTHs in living plant tissues. Further, we will test whether, and how, AGPs bind non-covalently to XTHs and/or to xyloglucans and oligosaccharide fragments thereof. The specific sub-hypotheses to be tested are listed in Section J. Although the project is targeted at crucifers because of their experimental convenience and because of initial successes with these plants, the potential outcome of the work is an ability to regulate more closely the growth and development of essentially any food, fodder or industrial crop by modification of the structure and expression of cell wall components.