The overall objective is to provide definitive new information on the specific biological significance of selected components of the plant cell wall. We wish to test the hypothesis that particular wall enzymes, expansins and polysaccharides play specific, definable biological roles in the living plant / especially roles in the processes of cell expansion and development in general.
Individual objectives are--
* devising novel high-throughput screens for wall-related enzymes [the Edinburgh Cell Wall Group has already developed a simple high-throughput fluorescence-based screen for xyloglucan endotransglucosylase (XET) activity, and we will now devise comparable screens for some of the polysaccharide synthases that produce wall-matrix polysaccharides, acyltransferases that add acetyl, methyl and feruloyl ester groups to nascent matrix polysaccharides, exo- and endo-glycosylhydrolases that potentially attack matrix polysaccharides within the wall, and acyl-hydrolases that potentially attack the ester-linked moieties of wall polysaccharides];
* devising novel high-throughput screens for expansins [using labelled substrates such as semi-crystalline [hydroxy-tritiated]cellulose, [hydroxy-tritiated]holocellulose, complexes between pure cellulose and labelled cello-oligosaccharides, and complexes between pure cellulose and labelled hemicellulosic oligosaccharides];
* assembling a collection of xenobiotics among which to seek inhibitors [the major sources being commercially available collections of natural products, glycosidase inhibitors, and herbicides, and also the Fry lab collection of organic chemicals which currently includes ~1600 compounds, many of which are in some way cell wall-related];
* screening the collection of xenobiotics on the novel dot-blot assays and identifying likely inhibitors;
* modifying the more promising inhibitors by attaching wall-related oligosaccharide groups [to achieve this, we will prepare a set of oligosaccharides that are themselves derivatised by introduction of amino, carboxy or oxo groups and then depending on the nature of any reactive group present in the xenobiotic, suitable oligosaccharide derivatives will be used such that an oligosaccharide/xenobiotic conjugate can be synthesised for inclusion in the screening programme];
* defining the effectiveness, specificity and phytotoxicity of inhibitors, including those with attached oligosaccharide moieties;
* defining the effects of specific, non-toxic inhibitors on the living plant, especially effects on cell expansion.
THE CHALLENGE: Growing plants have unique cell walls, indispensable for cell expansion, an aspect of plant growth utterly different from anything occurring in animals and microbes. Much was known about enzymes present in cell walls, and it was thought that the list of wall enzymes was more or less complete; however, we still cannot ascribe specific biological functions to specific cell-wall enzymes. Usually, genetics would powerfully reveal biological function: the gene for a wall enzyme would be mutated and we would observe the biological fate of the plant. Unfortunately, this approach is difficult to apply to cell walls because there are often dozens of genes in any given plant all encoding a family of very similar wall enzymes (‘isoforms’). For example, there are 33 isoforms of a wall enzyme called XET and it is not practicable to mutate all 33 of them. Our alternative approach is to apply foreign chemical agents (‘xenobiotics’) that block the activity of all isoforms of a given family of proteins. A xenobiotic inhibiting all 33 XETs would let us test critically the hypothesis that XETs (as a family) play a vital role in cell expansion. This ‘chemical genetics’ approach depends on discovering such xenobiotics: our 3-year challenge.
GOAL 1 — obtaining wall enzymes: First we identified rich botanical sources of the 12 wall enzyme families of interest, preferably supplied by a minimal number of different plant extracts. In fact, all 12 were richly provided by just 2 extracts (from alfalfa seedlings and parsley leaves). It was unnecessary to purify the enzymes because we required samples containing as many isoforms as possible so as to look for xenobiotics that simultaneously inhibit all isoforms of a given family.
A BONUS FROM GOAL 1: While conducting the enzyme survey across the plant kingdom, we discovered 6 new enzyme activities. One of these (MXE) was astonishingly restricted, occurring only in a single genus, Equisetum (horsetails). This genus is exceedingly isolated, having split from its closest living relatives over 370,000,000 years ago (well before dinosaurs appeared). MXE acts on an unusual wall polysaccharide (MLG), previously thought confined to cereals and related plants; but we found that Equisetum also possesses MLG. The novel enzyme, MXE, may have future agricultural potential if introduced into cereals, which possess MLG but lack MXE. A second new enzyme is currently under discussion for patenting, and 4 more new enzymes (see technical section) also came to light. The moral of this story is that cell wall enzymes are not a closed book and that plant biochemistry can still reveal new ones.
GOAL 2 — xenobiotic collection: We sourced 4,215 xenobiotics from commercial suppliers, our existing lab collection and a collaboration with Dr S. Cutler who had independently assembled a collection of plant-growth-modifying xenobiotics.
GOAL 3 — Devising simplified tests for the enzymes of interest: To make 50,580 enzyme assays feasible (see Goal 4), we devised simple, ‘high-throughput screens’ for each enzyme family.
A BONUS FROM GOAL 3: For devising these tests, we synthesised labelled substrates (‘tagged’ with an easily detectable radioactive or fluorescent group), which were not available commercially. Our laboratory is therefore now in a position to produce these substrates for sale. This is currently being discussed with Edinburgh Research & Innovation.
GOAL 4 — discovery of xenobiotics: We screened all 4,215 xenobiotics for effect on each of 12 enzymes. Of the 50,580 permutations thus tested, we struck 73 ‘hits’, i.e. xenobiotics inhibiting one or a small number of wall enzyme families. These 73 chemicals are now available for future experiments to assess their potential as agrochemicals and as scientific tools for a ‘chemical genetics’ investigation of the basic biological mechanism of cell expansion during plant growth.
(a)Thanks to the policy of assaying enzymes biochemically, we discovered six novel wall-remodelling activities [two ‘hetero-endo-endotransglucosylases’ (MXE, CXE), one ‘homo-endo-transglycosylase’ (trans-beta-xylanase), and three ‘exo-transglycosylases’ (trans-alpha-xylosidase, trans-beta-xylosidase, trans-alpha-arabinofuranosidase)]. Mainly by TLC-based assays, we identified optimal botanical sources and extractants for these plus 15 known wall enzymes.
(b)We synthesised labelled substrates and devised novel high-throughput screens for numerous wall enzymes (including pectin methylesterase, acetylesterase, feruloylesterase, MXE, CXE, polygalacturonase, xyloglucan-oligosaccharide-acting alpha-fucosidase and alpha-xylosidase, 1,3-beta-glucosidase, 1,4-beta-glucosidase, 1,4-beta-mannanase...). We also made progress towards an expansin activity screen using the novel substrate, [hydroxy-3H]cellulose.
(c)We assembled a collection of 4,215 xenobiotics, tested them in our high-throughput screens for 12 enzymes (= 50,580 assays), and obtained 73 ‘hits’, i.e. xenobiotics inhibiting specific wall enzyme activities. These xenobiotics are now available for assessing as potential agrochemicals and as tools to investigate the processes responsible for plant cell expansion.
Notes on output and impact added 3 March 2014:
A manuscript is currently submitted reporting the effects of >4000 small-molecule ‘xenobiotics’ on one representative cell-wall enzyme activity, namely xyloglucan endotransglucosylase (XET). A subsequent manuscript is also ready to submit, which will deal with xenobiotics targeting 11 other enzymes; this is being delayed during current negotiations with industry concerning the commercial application of the ‘hits’ generated. Additional manuscripts are also in preparation which will report the methodology of the substrate labelling and enzyme assays developed during the SCIBS project.
Concerning the xenobiotics that target XET, we are proposing that small molecules (xenobiotics) which inhibit cell-wall-localised xyloglucan endotransglucosylase (XET) activity would be valuable for elucidating its biological roles. We have developed a high-throughput screen for such inhibitors. Numerous plant extracts were first tested for XET activity by high-throughput ‘dot-blotting’ using filter-paper impregnated with fluorescent substrate, and by quantitative radio-assays in free solution. A parsley extract was selected because it produced uniformly fluorescent dot-blots that faithfully represented the extent of the XET reaction (enzyme concentration × incubation-time). This parsley extract was used for high-throughput dot-blot screening of the effects of xenobiotics on XET activity. Hits were authenticated radiochemically. Of 4216 small molecules tested by dot-blot assays on paper-immobilised xyloglucan, 18 inhibited XET activity, 18 promoted it, and 13 coagulated the enzyme. The strongest apparent promoters were anthraquinones and flavonoids. On radiochemical re-testing with soluble substrates, no xenobiotics convincingly promoted XET, but 22 inhibited it, including some that had been scored as promoters by dot-blotting. The strongest inhibitors were singlet oxygen (1O2)-generators e.g. riboflavin (IC50 29 µM), retinoic acid, eosin (IC50 27 µM) and erythrosin (IC50 36 µM). The involvement of 1O2 was supported in the case of riboflavin, whose XET-inhibiting effect was light-dependent. Other inhibitors were tannins, sulphydryl reagents and triphenylmethanes. The ability of some xenobiotics to promote XET activity in dot-blots (but not in free solution) may relate to the enzymes’ interaction with paper, a cell-wall-like surface. The compounds identified can now be tested in vivo, potentially revealing biological roles of XET, in a ‘chemical genetic’ approach.