Rydyn i'n gweld yn siŵn ni'n The chemicals that protect plants against pests and diseases attract herbivores, pollinators, seed dispersal agents. Of course we use them as flavours, fragrances, colours, scents, drugs for industrial biotechnology applications. They're a hugely valuable resource, but they're very complicated. My interest in this area started with a particular group of chemicals called triterpines, which are one of the largest and most structurally diverse types of specialized metabolite produced by plants. The triterpines share a common pathway with sterol biosynthesis, which of course is essential, because sterols are essential components of membranes and they're also hormones. Sterols are essential, they're part of primary metabolism, and sterols are made via the mevalonate pathway here, which goes to this precursor, 23-oxidose squalene, and 23-oxidose squalene is linear, but it can be cyclised by sterol synthases, in plants this would be cycloartanol synthase, to make the first committed precursor in the sterol pathway here, cycloartanol, which can then go on and be modified and turned into various sterols. Alternatively, this molecule can be cyclised to make a range of alternative products that come under the heading of specialized metabolism. Now there are some caveats around that, which we can come back to. So this is a fascinating molecule, and the enzymes that do this origami process are equally, if not more so, more fascinating. And these are just a few examples of the alternative cyclisation products that can be made from 23-oxidose squalene. And I'm going to focus in a little while on this one here, which is called beta-amarine. It's a pentacyclic triterpene. It's one of the most common triterpenes found in plants. So beta-amarine happens to be very common in plants, but it's also a precursor, oh sorry, before I say that, I've just mentioned that these are some of the alternative cyclisation products of 23-oxidose squalene. Actually there are an awful lot more of them out there, and these are just a few of them. So these are structures that have been reported from nature, and their structures are consistent with their being made by cyclisation of 23-oxidose squalene. So just with this very simple precursor, we're already getting a huge amount of structural diversity. And simple triterpenes have important properties in their own right. For example, oleonolic acid, which is the derivative of beta-amarine, the molecule that I just mentioned to you, has weak anti-inflammatory, anti-cancer activity, and there has been a lot of interest in trying to make improved versions of this that can be used for therapeutics. But of course a molecule like this is pretty difficult to modify chemically, selectively, because the scaffold has very little functionalisation to work with. So simple triterpenes are important, but in plants these molecules are often converted to far more complicated things. And here I'm just showing you a few examples of rather more complex molecules that are all made from that simple starting point, beta-amarine. And you can see that I've highlighted a few properties associated with these. So they all have this simple scaffold, but they have various other modifications. So they have oxigenations, they have sugars, here is an asile group, and you can see that there is a lot of diversity. So diversity in the scaffold, and then all of these further modifications, and there is a whole suite of enzymes that mediate this, can convert these molecules into all kinds of interesting bioactives. Very few of these can actually be purchased, and there are lots of problems with accessing them from nature because you have to access the plant material, they're produced in small quantities, they're often present as complex mixtures, there are a lot of issues with trying to get hold of triterpenes so that we can do systematic analysis to understand the relationship between structure and function. And you can see the properties listed here are diverse. This one, which I'm going to come back to, happens to fluoresce bright blue because it has an end methyl antherinolate group here. This is a very unusual property amongst the triterpenes, and as I will show you it's been extremely useful to us. And this molecule is produced only in the roots of oat, the genus avina, and it's antifungal. It also is phytotoxic, so it's a natural herbicide. And then you can see down here we have another triterpene produced by pea, called chromosome 1, and this, instead of being phytotoxic, is actually a plant growth stimulant. So we're really interested in understanding whether those molecules act antagonistically on the same pathway or whether there are different things going on. This molecule produced by legumes is associated with bitterness and antifedant properties, whereas this one from licorice is 50 times sweeter than sugar. And then we have various pharmaceutical properties listed here as well. So this is just a snapshot. This is a very, very small amount of the diversity that's out there in nature. And what got me interested in this whole area is this molecule here, which is called avinacin A1, and this is one of those stories of serendipity. And it started because I was interested in some papers published in the 1940s by a lady called Elizabeth Turner at the University of Oxford, who had seen that the tips of oat roots, avina, fluoresced bright blue under ultraviolet light, and she made extracts from these roots, and she found that there was a substance in there which was antimicrobial. And she called this root tip glycoside because she'd shown that it was glycosylated. At the time she didn't know the structure, which was subsequently determined, and shown to be this molecule, which has a beta-amarine scaffold, the fluorescent N-methyl anthronylate group here, and a trisaccharide chain. So this molecule is produced specifically by oat, so most of these specialised chemicals produced by plants are only produced by particular narrow taxonomic windows. So that's why they're called specialised. They're also often called secondary, but the term secondary is going out of fashion because they're clearly important molecules. And I was interested, so Elizabeth Turner in the 1940s proposed that this molecule might be protecting oats against attack by soil-borne pathogens. And I embarked on this somewhat reckless set of experiments to address this because at the time people were really interested in molecules produced in plants in response to pathogen attack. And this molecule is just sitting there in the soil. It's produced as part of normal growth and development. And nobody had asked at the time whether preformed chemicals might be important in protecting plants, and to me that seemed like an obvious possibility. So we took diploid oat and we treated it with a chemical mutagen, and we simply screened by putting germinating oat seedlings onto a UV transilluminator. We looked for seedlings with reduced fluorescence, and this is one of them here. Clearly this molecule is very complex. We expected to find many genes involved in its synthesis. At the time that we started this, nothing was known about triterpium biosynthesis for any plant species. Oats are very unusual amongst the cereals and grasses in making antimicrobial triterpenes, whereas the dichotoledinus plants make a huge array of triterpenes. So this was an unusual situation in the grasses. And people were interested in why oats were making this and other cereals such as wheat and barley and maize were not. So we did this mutagenesis.  In fact, we have nearly 100 mutants in this very complex pathway. And then we tested the mutants to see if they were compromised in disease susceptibility, and the answer was very clearly yes. So here is the wild type oat, and this has been challenged with a soil-borne pathogen that causes massive disease losses on wheat, and it's resistant. The mutants all show severe lesions and are clearly susceptible. So we had addressed Elizabeth Turner's question, and we had shown that this molecule appears to be involved, or it is involved, in protection against soil-borne pathogens. And we would have stopped there because diploid oat is not a model species by any means. It's not a rabidopsis. And this is a very, very complicated molecule. But the reason why we kept going was because the genetics started to tell us that something very strange was happening. The loci that we defined by mutation but had not yet characterised were not cosegregating when we did genetic analysis. They were sticking together. And this suggested that the genes for this very complex pathway were genetically linked in the genome. And many years on, we've now characterised all of the genes in the pathway. Here I'm just showing five. This is the gene for the first step, the second step. These are three genes for the ASR group. And these genes are physically adjacent. Those of you who work on bacteria might think this is really funny because this is 400 kilobases here. This is a big distance. But actually in plants, this is a gene-rich region. And there are no other obvious genes in between the genes that we find. And I should emphasise that these genes all encode entirely different types of enzymes. This is the oxido-squaling cyclase that converts 2,3-oxido-squaling to beta-amarine. This is a P450 that introduces modifications to beta-amarine. This is an ASR transferase, a sugar transferase, a methyl transferase. So this is not a tandem duplication event where we have an array of genes that have been duplicated. And I can also tell you with confidence that this region has not originated from microbes. It is not a horizontal gene transfer event. And I can further say, and there is a lot of evidence for this, that this cluster has evolved since oats have separated from other cereals and grasses. So something very strange is happening. Now when we started this work, the general understanding was that genes for the synthesis of specialised metabolites and for other multi-step processes were scattered around the genome. But here we have something that looks a little bit more like a bacterial operon. It's not an operon because the genes are transcribed separately, but nevertheless they are physically clustered. Together they make this molecule which is required for plant defence. The molecule, if you take a cross-section of an oat root, is in the epidermis. So it's in the right cell layer to provide protection. It makes sense. The genes are expressed in the roots but not in the other parts of the plant. And when we do messenger RNA in situ hybridisation, we can see that, in fact, they're also expressed in the epidermis. So these are longitudinal sections of the root and this is where the signal is. So we have physical clustering and co-expression. This is a recently evolved pathway. These genes have somehow been recruited from existing components elsewhere in the genome and assembled, I'm choosing my words carefully here, assembled into a cluster. Now we know a lot more about this pathway, which I won't go into the details of. We've characterised all of the genes and enzymes now. We haven't published on all of them. We know that the blue fluorescent molecule ends up in the vacuole. We know where a number of the proteins are. We do know that these proteins do not exist as a single multi-protein complex because we know, for example, that the acyl transferase is in the vacuole, that the early steps are likely to be on the cytosolic face of the endoplasmic reticulum and that the other intermediate steps are probably cytosolic. And here is the molecule in the vacuole. So one of the very ambitious projects that we have is to see, having learnt what we have from oats, can we take these genes and engineer them into wheat and into other plant species to see if we can make antimicrobial triterpines, not necessarily the blue fluorescent one exactly, but to make triterpene scaffolds and modify them to give antimicrobial compounds. And if we can do this, then perhaps we can prevent this dreadful soil-borne disease of wheat, which causes hundreds of millions of pounds of loss in yield in the UK alone per year. So this is a very ambitious project. One of the things that surprised me when I first started thinking about plant engineering is that despite all of the effort that has gone into this area since the 70s or 80s, there are very few promoters available. And there are certainly very few promoters available that we can have confidence that they will drive co-ordinate gene expression if you want to express multiple genes in the same tissue at the same time. There's still a big challenge there. And we found something rather surprising, which was although when you look at our gene cluster, we can come to the reasons why genes might be clustered and I'm very interested in your views on this. But clearly one explanation or one partial explanation could be that physical clustering enables a higher level of regulation of the gene expression at the level of chromatin and potentially also higher nuclear organisation. However, if we take the promoters of these genes out of this region and we hook them up to a reporter gene and we put them into other plant species, they work. So this is oat and here you can see fluorescence in the root tips as I've shown you. What I haven't shown you is we also see fluorescence in the lateral root initials. So this is the pattern of expression. If we hook the promoters up to a reporter such as Gus, we get blue staining where we see expression. So in diverse plant species such as a rabidopsis and rice and legumes, this is just with the promoter for the first gene in the oat cluster, but it's true also for the others. We see Gus expression in the root tips and the lateral root initials in these diverse plant species. So remember that this pathway has evolved recently. It's specific to oat and so this provides a further conundrum. It looks as though that the promoters for the oat cluster have somehow plugged into some sort of ancient, highly conserved root development process which is conserved across the monocots and the dicots. It's also very useful because it means we can use these promoters in other plant species and here they are in wheat. So this is really nice. We now have a set of 11 promoters that we can use to drive the expression of multiple genes in wheat roots. That's a really useful resource. We still don't quite understand how it works. Coming back to the triterpenes, I mentioned that 2-3-oxidose squalene could be cyclised. It can be converted into all of these different cyclisation products by enzymes known as 2-3-oxidose squalene cyclases and I showed you examples of all of these diverse scaffolds and we now know, oops, very many years on, we now know a lot more about the enzymes that make these scaffolds. We still haven't characterised very many of them but these are some examples. The oat one is up here. Interestingly, dicots make beta-amaran in different ways and then there are other enzymes that make all sorts of diverse scaffolds. So it's a very interesting group of enzymes because they take one substrate but they're able to convert it to all of these different products. Most of these enzymes are specialised, some of them make multiple products. We're beginning to learn about the cytochrome P450 enzymes that are able to oxygenate these scaffolds. So this is beginning to provide us with tools that we can use to selectively modify scaffolds in different places and these are all beta-amaran modifying cytochrome P450, not just from oat but from a whole range of other plant species. So we're putting together now collectively a number of groups around the world have discovered a lot of genes and enzymes for the synthesis of triterpene scaffolds and their modification and we're putting together a toolkit that we can use to make sweets of structural variants of these molecules. So these are beta-amaran modifying P450s. There are also P450s available now that make other scaffolds which we and others have characterised and you can see that we're building up a very powerful set of resources here for the modification of scaffolds which can then be further modified enzymatically or of course they could be modified using chemistry. And we're beginning now to learn also about the sugar transferases because we've had to work our way through the cyclases and then the P450s to get to the next step which is glycosylation and there are now quite a few sugar transferases that put sugars onto various positions around the scaffold and of course glycosylation also changes the physical and the biochemical properties of these molecules quite markedly. One of the things that has been very helpful to us, I mean there's a general feeling out there that working with plants is extremely slow and when you're doing genetics they know it is but one of the things that's really accelerated what we have been doing is this very nice transient plant expression system developed by my colleague George Lomonossoff at the John Innes Centre and what this involves is a very simple system where you can take your gene of interest in this case GFP and George accidentally discovered a number of years ago through his work on Calpimazec virus that if you have a little bit of Calpimazec virus sequence from the RNA2 gene of Calpimazec virus inserted in front of your gene of interest this gives a massive elevation in the amount of protein that's produced and this effect is post transcriptional and so if you have your construct with your Calpimazec virus sequence here and a suppressor of gene silencing P19 put these into agrobacterium the workhorse that we use for plant transformation you just squirt your agrobacterium into the leaves and within five days or six days you have in this case very clear expression of green fluorescent protein and the levels of protein using this system because of these sequences are massively elevated and so we wondered whether this would also work for the molecules that we're interested in and so we made constructs and we did this expression in this Nicotiana Benthymiana plant which is particularly amenable to transient expression and here we've got GC traces and this is an extract from an empty vector control leaf this is an extract from a leaf expressing our beta-amarine synthase and this is a peak of beta-amarine so this is the black scaffold here and then here we have the second step in our OAT pathway which is a very interesting cytochrome p450 which we knew modified beta-amarine but we weren't sure exactly how and when we co-infiltrate the genes for these two constructs the beta-amarine peak goes right down and this peak comes up and we were able to easily get enough to purify and determine the structure using NMR interestingly although I won't go into this in any detail this p450 is able to modify two rings on this structure it's a very interesting enzyme so this then was a proof of concept and we've now gone on taking our triterpine toolkit you'll hear more from Jim about the common plant syntax and the ways in which we've been getting our DNA synthesized and formatted so that we can mix and match and exchange with others so we have a toolkit of genes and enzymes for triterpine synthesis modification to make known and novel molecules and we can now play with these we put them into the the nicotiana leaf expression system and within five or six days we get our answer we know which enzymes will do what so this is very very rapid very quick very powerful and we've been using it to assemble multiple steps in in the synthesis of triterpines to make very complex molecules and we've been able to easily make several hundred milligrams which is useful it's very useful for doing bioassays and recently somebody in the lab has now made a gram of beta-amarine so we can go up to gram levels the question now is can we go down to high throughput robotics and you'll see how that can be even more powerful fairly soon now i mentioned this phenomenon of gene clustering and i said it was unusual it was surprising and at the time we found the oat gene cluster there was only one other example of a gene cluster for a specialized metabolic pathway implants and that had been reported by Alfon's Gil in Munich who was working on maize on molecules known as benzoxazenoids which are also implicated in plant defence that was the only other example that was about 15 years ago now but the sorry the general view until quite recently has been that genes for multi-step pathways are dispersed around the genome and the anthocyanin pathway in maize is a great example of this and the genes for anthocyanin biosynthesis are on different chromosomes in maize and are used to follow the segregation of genes in genetics it's well known you could say well are anthocyanins really specialized because plants all plants make anthocyanins carotenoid genes similarly are dispersed carotenoids are also very widely distributed nevertheless there are examples of other specialized metabolic pathways for example for glucosinolates in brassicas that are not clustered so we wanted to find out how common this clustering phenomenon might be there was the original maize cluster the oat cluster so we went to a rabidopsis because at the time there weren't many plant genome sequences available and the rabidopsis genome was very high quality and we did a very crude mining experiment we simply looked for genes that were predicted to encode two three-oxidosequalene cyclases so genes that look like they might be able to take two three-oxidosequalene and turn it into one of those cyclic products that I've mentioned and in the rabidopsis genome there are 13 genes that belong to that family one of them is required for the synthesis of cycloartanol which is as I said the precursor for essential sterols but the others all make diverse products and that's been shown in yeast so they're not involved in essential sterol biosynthesis this is one of those genes when we look around that gene in the genome we see that its neighbors are genes that are predicted to encode two very different types of cytochrome p450 that have not arisen by tandem duplication they're very different and anacyl transferase and we also saw that these genes were very tightly co-expressed in the available gene expression data so this looked like a candidate metabolic gene cluster and so Ben Field who was in the lab at the time did some very careful analysis using mutant lines that were blocked in these various steps and also using overexpression lines and silenced lines and biochemistry and he was able to show that these genes did indeed form a pathway and this was we called it rather mischievously the first example of an operon-like gene cluster in Arabidopsis it's not an operon as I say but the point is these genes are not tandem duplicates they encode steps in a pathway they're co-expressed so they have operon-like features so we and others have then gone on to to predict and validate other metabolic clusters in Arabidopsis and there's a second one that we found the first one was for the synthesis and modification of Thallianol which is a triterpene and then another one for the synthesis and modification of manarol another triterpene I should say at this point that although the oat of venosyn pathway and the Arabidopsis triterpene pathway which I've just shown you are both triterpene pathways there is compelling evidence that these pathways have evolved independently of each other and if anyone wants to know more I can tell you afterwards so this would appear then to suggest that triterpene pathways may be predisposed to clustering we found a second triterpene cluster in Arabidopsis the manarol cluster and through careful analysis and looking at phylogenetics and intron exon patterns we were able to put together this model which may be a little bit too generous I mean we it's possible there was an ancestral gene pair that that was the foundation for both of these clusters but we can't be sure of that and this is contained in oxido-squating cyclase and potentially also a particular type of cytochrome p450 so conceivably then this duplicated but nevertheless what has happened since then is that other genes have been recruited into these regions and these clustered pathways both make entirely different products so this is a possible scenario for the ways in which in Arabidopsis at least these clusters may form now since the maize and the oat story and the Arabidopsis work others were discovering other clusters in other plant species work from Ruben Peters lab and Professor Yarmone's lab in Tokyo had led to discovery of clusters in rice again for the production of defence-related compounds, di terpenes and this figure which you won't be able to see because it's too small is from a review that we published at the beginning of last year and this shows clusters from a growing number of different plant species for different types of chemical excuse me and this this just gives you an example of some of those so here we have the the oat compound so these molecules are all produced by clustered pathways here is the maize compound Arabidopsis there have been clusters reported for the synthesis of anti-nutritional compounds in tomato medicinal drugs in poppy these are the rice di terpenes clusters interestingly the cyanogenic glycosides which have traditionally been sought of as the most ancient and highly conserved group of plant natural products and these are sporadically distributed across the plant kingdom it turns out that those clusters and this is work from a group in Copenhagen sorry those pathways are clustered in lotus in sorghum in cassava but importantly those clusters have arisen independently so independent evolution then of genes that give rise to cyanogenic glycosides in diverse plant species so the reason for clustering now depending on your background you may have different explanations for this this is clearly a non-random organisation of genes in the genome plant genomes generally contain around 30,000 genes so to have genes right next to each other that are not tandem duplicates that are delivering these beneficial pathways is definitely not random and as I mentioned earlier physical clustering has the potential to facilitate higher level regulation at the level of chromatin at the level of nuclear organisation and I've shown you that the the genes in the oak cluster have this very very tight expression pattern in Arabidopsis this is in silico gene expression data these are the four genes of the thallionol cluster and you can see their expression patterns across different tissues is very similar when we get to the edges to genes that are not involved in this pathway the expression pattern is different so these are windows of co-expression we showed some years ago using DNA fluorescence in situ hybridisation with the oak cluster that if we take probes for the gene at the first gene in the pathway and the second gene in the pathway in green and red here so you can see this is looking at DNA now you can see that in the epidermis these signals are quite strung out they're quite extended and of course the pathway is active in the epidermis whereas in the cortical cells where the pathway is not expressed the signals are more discreet and it was possible to actually quantify these these differences so this is descriptive but it suggests that chromatin decondensation is associated with expression of this pathway in oat. We've gone on to do a lot of work in Arabidopsis where the resources are much better for looking at chromatin modification the the effects of various modifications on cluster expression and a picture is emerging now both in Arabidopsis and from what we've been able to do with other plant species where the resources are available so in Arabidopsis for example genome-wide chromatin immunoprecipitation has enabled regions to be identified that are marked with a histone modification this is histone 3 k27 trimethylation and this is a usually a repressive marking and these clusters have a very very discreet and localised marking of this h3 k27 trimethylation and this is generally installed by the polycomb complex so this is associated with inactivity but we also see that in tissues where clusters are active we have exchange of the histone 2a protein with histone h2a z which again has been associated with poisoning and with with readiness of genes for example in yeast to enable rapid expression in response to environmental change so we still have a lot to learn about this but i should also say that while in animals regions of contiguous marking of genes with h3 k27 trimethylation is well established in plants it isn't so there has been a lot of work in plants on polycomb mediated regulation of gene expression most notably in the context of plant development but these genes are generally isolated they're not contiguous and it transpires that we can use these markings as an additional tool to try to mine genomes to discover new pathways and here you can see these are the genes in the thallianol pathway in arabidopsis and this is the histone 3 k27 trimethylation marking which is very clearly significant and pronounced and we can actually go through data from the arabidopsis genome we can look for windows of co-expression we can also look for windows of h3 k27 trimethylation in tissues where the pathways are not active and we can use that to find new candidate pathways similarly i mentioned that h2a z this alternative histone 2 variant is associated with pathway activation and we can use mutants to look for windows in the arabidopsis genome where we see a very clear effect on transcript levels of cluster components so this is the thallianol cluster this is in the wild type line this dotted line but in a mutant that is lacking h2a z you can see that the transcript levels go right down and there's this very discreet window this is very striking similarly for the manarol cluster and this is a new cluster that we've recently discovered and that others have also done some work on and this is a much larger cluster and it's very very pronounced so we're beginning to learn about the features of these clusters not only in terms of the genes within them but also these kinds of markings and this can all be fed in to what we hope ultimately will turn into a machine for mining plant genomes for discovering new pathways and chemistries so the arguments for clustering i've talked about co-expression i've also told you that these clusters have either been shown to make compounds that provide protection against pests and pathogens or they're likely to have some role in survival in the environment so it's generally accepted that these specialised metabolic pathways are serving a useful purpose in nature so if you have assembled good combinations of genes that together are able to make a protective molecule presumably once you've done that you want to co-inherit that gene set together in order to be able to continue to make the molecule so that leads to another theory that has been proposed which is the co-inheritance theory on top of that we also know that in some cases if we interfere with these pathways with mutants or with overexpression of pathway genes then we get elevated levels of intermediates accumulating and this can have clearly detrimental effects not always but in some cases so in OAT for example failure to add on a glucose to the trisaccharide chain of the triterpin leads to a very sick root phenotype and here inside the wild type this is normal wild type root cell this is inside the mutant the epidermal cell layer is very distorted there are these big membranous sacs and this is callows chaining an accumulation of callows in this way is typical of a response to toxicity and in fact if we combine this mutation with a mutation in the first step in the pathway we lose the ability to make the intermediate and we restore the normal root phenotype although clearly we don't make the molecule so this is a toxic intermediate effect similarly in a rabidopsis if we mess with the thallianol cluster or the manorol cluster we can have these clearly detrimental effects on plant growth so if you interfere with clusters you not only lose the ability to make a protective molecule you can also generate molecules that are very bad that cause serious effects on growth and development so this is very simplistic and as i said it depends on your background which of these you might jump to and for the population genetics in the audience geneticists in the audience there's another level obviously so co-expression co-inheritans toxic intermediates these are not mutually exclusive arguments and they're also not the full story and certainly we don't always see toxic effects when we interfere with pathways and i think we can learn something so i'm victor's comment at the beginning of the meeting about separating out where do biological systems biological objects come from and how do they work to me that's a bit of a blur i mean you can't recreate evolution obviously but i think we're getting glimpses into things into what might have happened in terms of cluster assembly and how clusters work through these kinds of experiments so this issue then of how do these pathways assemble evolve is a bad word in this certainly in this forum how do they assemble and the answer is we still don't really know i've shown you a model from Arabidopsis we have a gene encoding the first step in the pathway which makes the scaffold or at least is the first committed step in the pathway and then we have genes encoding enzymes that modify the product of the first enzyme and we call these tailoring enzymes by analogy with microbial systems and these are things like cytochromes p450 sugar transphases acyl transphases methyl transphases so often the gene for the first step in the pathway as i've shown you for the triterpenes has almost certainly been recruited from primary metabolism in the case of the oat and the Arabidopsis triterpenes from the sterol pathway but then it has diverged it has acquired a new function and also a different expression pattern somehow then this gene has ceded the formation of a cluster and again i'm using my words very carefully here but there is lots of evidence to indicate that these clusters are forming de novo in plant genomes and this is where if the mathematicians have any ideas i would be really grateful to to know them so i've shown you examples of toxicity when you accumulate into medias but also some other very subtle effects and this is one of them so going back to our oats this is the the wild type oat line this is a mutant that is blocked in the first step in the pathway it's not able to make the beta-amarine that is the precursor for the the rest of the pathway at least not in significant quantity this is a late pathway enzyme this is the acyl transferase these roots all look normal we noticed and we're growing these seedlings on big square petri dishes and we had to do that to see this effect because initially we were growing them on small petri dishes we kept thinking the roots of this mutant look shorter but it was a bit variable and here you can clearly see that these roots are shorter and these mutants are blocked here so they're accumulating 40 fold more beta-amarine than the wild type so this is a beta-amarine mutant that is blocked in the first step in the pathway there's still a basal level there's a very low level of beta-amarine detectable in the wild type and a trace detectable here but these mutants are accumulating 40 fold more beta-amarine which as i said is a common plant metabolite and when we look closely at what's going on in these roots these are the root hairs this is the wild type these are mutants blocked in the first step and the roots look normal these are our sad two mutants the mutants that are accumulating beta-amarine and the roots are super hairy these are two independent mutants this is an intermediate mutant that is partially blocked so somehow accumulation of elevated levels of beta-amarine is triggering this super hairy root phenotype and when we looked at this very closely we saw something really interesting so this is patterning in the wild type root epidermis this is before the root hairs emerge we're looking at the the meristem the long gray cells are the cells that will not give rise to root hair cells the pink cells are shorter and they are the cells that will give rise to root hairs in the wild type we can detect low levels of beta-amarine in the mutant we detect as i say a lot more and what we're seeing is actually a change in cell specification this is really important this is happening very early on when the cells are deciding what they're going to be they're being told before they form that they're going to be root hair cells and this is why we have this super hairy root phenotype so this is a bit more like a plant growth hormone effect so when you start to think about all of these things we're seeing the things that intermediates do trying to make sense of it it's a big challenge but there are definitely some really interesting things coming through this so super hairy roots at some point in time may actually have been an advantage maybe you take a cyclotonal synthase gene you duplicate it it acquires a new function the ability to make beta-amarine it's expressed in the roots and maybe that was a good thing and we're now looking to see whether these mutants have enhanced biotic and abiotic stress tolerance as an aside we also see interesting more subtle effects with other situations as well so for example i've shown you about toxic effects with the thallionol pathway in a rabidopsis but we also see some more subtle effects where if we accumulate elevated levels of thallionol in the roots we actually see longer roots if you want to make longer roots and make a fit of plant that's quite a useful thing to know about and in legumes which form symbiosis with nitrogen fixing bacteria there is a different triterpene called lupiol and there is a lupiol synthase that is expressed in nodules when we silence it we see a window of clearly elevated nodule formation so lupiol appears to be suppressing nodule formation so we don't understand all of this but i'm just saying the stuff going on and it gets more complicated and so these these are just more examples of different effects that we can see when we tinker with clustered pathways so just before i finish i want to tell you a little bit more about what we've been doing in a more bigger picture way because i hope you can see how once this is all sorted out and automated this could be incredibly powerful the whole clustering phenomenon and one of the things that we've been doing this is with Alex Butanov who is a bioinformatician based in Russia is taking multiple plant species in this case 17 species for which the genome sequences are available we focused on the terpene family so the terpenes include the triterpenes but also a whole load of other types of terpenes that i won't go into but it is a massive family of diversity in plants and what Alex did was he mined these genomes for all terpene synthase genes so these are the engines of generating diverse scaffolds and he also mined them for all cytochrome p450 genes and then he asked using a sliding window approach how often he found a terpene synthase gene in proximity to a p450 gene relative to what would be expected by chance and the answer was very often and so we've classified all of the terpene synthase genes according to the nomenclature and all of the p450 genes and what we can start to see is that there is a clear skewing there's a clear tendency of p450 genes to be physically linked within 50 kilobases which implants is close to terpene synthase genes and we can also look at the patterns that are emerging in terms of the types of cytochrome p450 genes that tend to be associated with terpene synthases and when Alex did this he found the known examples of terpene synthase genes that have already been reported I've told you about the di-terpene clusters from rice he also found the steroidal glyco alkaloid cluster from tomato the arabidopsis clusters and so on but also a whole bunch of other candidate clusters one of the things he did which was very interesting in terms of trying to get more insight into how these pathways evolve was he took a couple of the major pairings the major groupings of terpene synthases and p450s and this is a little bit just bear with me this is easy to understand once you get it so he took these pairs of terpene synthases and p450s and he did sequence similarity searches so he took a terpene synthase within a pair and asked which one was the most closely related terpene synthase in the other pairs and then he did the same for the p450 partner of that terpene synthase and what he found was something very interesting which was that in the dicots the terpene synthase that that when you found the the one that was most closely related to it its p450 partner was also most closely related to the p450 partner of that terpene synthase in other words that's suggesting an iterative duplication of pairs of terpene synthases and p450 genes that was not the case in the monocots something strange is happening there so it's as if there is a micro-sintony in the dicots and this is kind of the opposite way round to the way that it's normally thought of i've spoken to a lot of genomics experts and they they don't know how to explain this and we're only looking at this on the basis of terpene synthase is not the whole organisation of the genome but it suggests that there is micro-sintonic duplication in the dicots and that would be consistent with our Arabidopsis hypothesis for the thallionol and manorol clusters but in the monocots it's all chopping and changing and de novo combinations he also looked at all of the the the genes within these pairs and compared them to genes across the genome looked at the frequency of transposable elements in the flanking regions because you might expect that transposable elements would play a role in moving genes around and as we might expect he found that there were significantly more transposable elements in the flanking regions of the genes within these pairs than in the ones that were not paired and we then went on and took some of these new candidate clusters that Alex had found and we validated them experimentally and this is work with Reuben Peters in Iowa so this was a predicted cluster for di-terpene synthesis in Caster this was another cluster in Arabidopsis again for triterpene and this is a cluster from Cucumber so again it's more grist for the mill it's more a very powerful way of finding new chemistries so in summary I'd just like to say that I've talked about examples of these metabolic gene clusters implants for the synthesis of diverse chemicals we don't yet know what the balance will be of pathways where the genes are dispersed relative to those that are clustered nevertheless clustering is very useful for mining genomes to discover new pathways and chemistries we can look at these clusters to try to understand what their defining features are and to understand how they work and then we need to know whether we can edit them down we don't know whether we can to make minimal gene clusters which will make it much easier to move these pathways around certainly in plants and make synthetic gene clusters and one of the things we've done for example is to take the promoters from the OAT cluster which you will remember are expressed exquisitely in the root tips and we've used those to drive the expression of a three gene pathway for the synthesis of cyanogenic glycosides in a rabidopsis roots and this opens up opportunities to start to tailor the risosphere by making designer chemicals implant roots and with that I'd like to finish and just draw your attention to the many people who have contributed to this work and also the funding sources and I think you'll hear more about open plant from Jim in the next talk thank you


With action and suspense out of the Old West comes the most famous hero of them all, Hopalong Cassidy, starring William Boyd. The ring of the silver spurs harrows the most amazing man ever to ride the prairies of the early West, Hopalong Cassidy, the same hoppy you cheer in motion pictures, and the same California you've laughed at a million times. Raw courage and quick shooting have built a legend around this famous hero. Hopalong is a name to be feared, respected and admired, for this great cowboy rides the trails of adventure and excitement. William Boyd as Hopalong Cassidy and Andy Clyde as California. Well, Hoppy, what about our story? We call this one the cold country. All the icy bitterness of the North had come looking across the Dakotas, and the town of Lode Hill was being sealed off by the worst blizzard of the winter. Even at that you felt you could find excitement if you went looking for it. Lode Hill had been a sleepy place last time I'd seen it, but that was before the gold strike. Now it had 10,000 people, quite a few saloons, and a hotel with a fancy heating stove in the lobby. This is where I'd taken a stand, within a few inches of that stove, where I could turn myself slowly, like a hen on a spit. Some weather we should have stayed in Arizona. Come on over here, California, solid comfort. You still want to see loop rangers? I want to see them. That's the reason we came north, isn't it? Then you'd better forget the solid comfort and come out into the fresh air with me. You found Luke? That's right, but he's in some kind of trouble, and he's fixing to pull out of town. In this storm, where is he? Livery's table, just down the street. He was saddling up when I left him. Come on, we'd better get over there in a hurry. Oh, boy. Oh, take it easy. You! All right, stay where you are there. Myra, you've got no idea how close you came to being... I know, but I had to see you. Joey Edmund says you're leaving and that you want to take in Nettie. That's right. Luke, don't leave. I've got to go, Myra. I've hung around too long now. But I'm grateful to you for coming here. Luke, Jane was my sister, but I know what she was like. She was that way with all of us, cruel, unfair. She's dead, Myra. I don't want to talk about it. Luke, don't go. Stay here and fight it out. I've thought it all over. Who's there? What do you expect, your lopiered lobo? California and Hoppy. Oh, it's great to see you. Ah, that goes double, but... Oh, excuse me, Myra. Myra, this is Apollon Cassidy in his pal, California. My sister-in-law, Myra McKenna. Howdy. Hello. That's a pleasure. And maybe you can tell me why Luke wants to hold up in a livery stable. The kind of weather we're having, this place doesn't fit for anything but storing ice. I'm not going to be here long. I'm heading out. Ah, that's great. All the way back in Arizona, I hear about my pal Luke striking it rich with a goldmine. So I write up here to congratulate him. And he won't even stick around for a celebration. I have to go, Hoppy. I don't have a choice. California says you're in some kind of trouble. He called it. I thought you might like to talk about it. Not much to say. My wife is dead. Happened last night, somebody shot her. Any minute, now they're going to tag me for it. But you didn't do it, Luke. Who knows that? Who'll believe it? I believe it, Luke. Me too. You can't run out on a thing like this, Luke. It's just like saying you're guilty. If I stay, I won't have to say I'm guilty. There'll be others who'll say it. Yell it, scream it. I got enemies around here. We all have enemies. And we have friends. It's a stacked deck, Hoppy. Me and Jane, we had a bad fight yesterday right in front of a lot of people. The only chance I've got is to get out of here fast. I'm just waiting for Joey Edmonds to bring the boy.  I'm not leaving him here.  Oh, Luke. Luke, listen to me.  You can take care of yourself. You might last out on the plains in this weather, but a six-year-old boy... It's no use, Hoppy. I've made up my mind. I'm leaving here with Nettie tonight. Get that door shut! Give me time, Belu. Give me time. Sot makes a piano sound as though he played it with his feet. What do you expect out here, Grand Opera? I told you before, Drag. Don't ride me like that. Any news from the coroner's jury? Still arguing. Fools. She was murdered and he did it. When I get through with public opinion...  What do you mean? Just talk to young Shafter. Seems to have it that Granger's taking his kid and skipping town. Why didn't you tell me this before? Where is he? Shafter's stable. He's waiting there for Joey Edmunds to bring the kid to him. Well, move. Take some of the boys. Get over there. What do we do? Burn them down? You leave him alone. I'm gonna see that stiff-necked reformer go to trial. I'm gonna see him convicted for his wife's murder and I'm gonna see him hang for it. So? What do we do? Leave him alone. He just got the kid. That'll keep Granger in town. We return to Hoppillong Cassidy and the cold country. In Shafter's livery stable, Hoppy and Myra McKenna are still talking to Luke Granger, urging him to give up the idea of leaving town. Stay here, Luke. Let California and I get all your friends together. Stay in trial and come out clean. That's the only way to do it. He's so right, Luke. You have to think of Nettie as well as yourself. Think. It's all I've done is think. You ought to know I don't water run. Everything I've ever worked for is in this town. And the kid. How do you think I feel about yanking him away from everything he knows and cares for? I've been doing so much thinking I'm just about local. But where would you go? What would you... What was that? Don't try anything, any of you. Get Max and Charlie in here. Okay, Drake. Sounds as though you brought along quite a crowd. I don't need a crowd. Any sound of that kid out there? No sign yet, Drake. Kid, what's that about a kid? Take it easy, Granger. You just want your boy, that's all. Oh, no, you... I told you to take it easy, Granger. Find anything else and I'll blast your other hand. Luke, is it bad? Nothing I can't handle here. Let me wrap it up with a scarf. Who is this fellow, Luke? Drag Moorley. Works for Parkwell Luke. Drag Moorley. That's right. I'm known around here. Oh, you're known far and wide. Rapid man with a pistol. And if you're smart, you'll keep that in mind. I think there's someone coming out, Drag. Might be the kid. Good. Everything else working out just right. You're too sure of yourself, Moorley. Yeah? How'd you make that out? It's pretty dark in here. And even though you got the drop on the... You can't see everything. My, you... There he's gone. You shot it right out of his hand, coffee. Hold it, Moorley. Go for that other pistol and I'll put the next slug in your chest. You scare me to death. Just don't try anything. Was that the boy, Luke? No, it must have been someone passing by. Who are you, anyway? The name is Cassidy. Hop along, Cassidy. Cassidy, huh? That's a big name. Too bad we had a meeting here. But there'll be other days. What do you want from me now? You can leave. Just make sure you stay away. And tell Baloo that from now on we'll be ready for any move he makes. You don't think that's gonna bother Baloo, do you? You'll hear from us again. Come on, Ed. How's the hand, Luke? Oh, I've had worse. If you still feel you want to pull out, I'll go with you. You might need help with the boy. I imagine California will come, too. Well, you know I will. Maybe I've changed my mind about leaving. Maybe I figure out a stay and make a fight of it. Oh, Luke, I could cry. Gotta wipe my glasses. They're all fogged up again. Could be a lot better for the boy, Luke. All right. I'll stay. I'll go looking for Bat Kingman right now. Bat Kingman? Tell him, Marshal. I've given myself up. Well, Mr. Kingman? Never thought I'd see the dad have to shut a cell door on Luke Ranger. I'm glad he turned himself in. Makes it a lot easier for me. How about the boy? I'll take care of Neddy. He'll never hear a breath of this if I can help it. Jane was my sister, but she never brought anything but trial and tribulation to Luke. Tell me something, Marshal. Why should Bart Baloo hate Luke as much as he seems to? Baloo heads the gambling element here. Luke loved this town long before gold was ever found and he wanted it kept clean. He's been the head of a reform movement we started recently. Oh, that's it. I've been urging Luke to stay here and face it. Now I'm beginning to wonder what kind of trial he'll get. I don't know, Cassidy. I don't know. Where are you going, Hoppy? To see Bart Baloo. Might be worthwhile having a talk with him. Hello, Baloo. Well, it isn't Hop along, Cassidy. Deal his choice here, Cassidy. Not this time. Just wanted a word with you. Go ahead. I happen to be a friend of Luke Granger's. From what I hear, he's going to need all the friends he can find. I'm interested in seeing he gets a fair trial. Aren't we all? Oh, I've passed, gentlemen. I'm interested enough to make it rather personal. Yeah? Why tell me all this? I ran into a palier a while ago. Dragged morally. Oh, yeah. Shot a pistol out of his hand, didn't you? In the dark. I might have been lucky. Then why not congratulate yourself and fade out of the picture? Meaning, uh... You can't always depend on darkness when you run up against drag morally. That's probably sound advice. Sure you won't draw cards? No, I'm kind of tired. I kind of just get to the hotel and turn in. You're in luck. Storm's just about over. See you later, Baloo. It's a rough town, Cassidy. Take care of yourself. We're cold out here, uh, waiting. Let's go get some rest. Gonna be a long hike back to the hotel through the snow. Baloo was right, though. He said the storm was over. Yeah? Uh, what else did he have to say? Told me to take care of myself. Well, now weren't that nice of him. There's something funny... About what? That saloon. Yeah? Doesn't seem to be doing much business tonight. Ain't nothing funny about that, not with this kind of weather. The weather was a lot worse last night and the place was rocking. Uh, yes, that's right, but, uh... Yeah, there's a couple lanterns bobbing up and down with the feed store and three or four more moving around near the hotel. Lanterns? Oh, now I see them. And there's another bunch coming in from the other end of town. Well, the more light the better. Might stop there. California. Hey, get down. What do you think you're doing? That's saloon. No wonder it didn't have many customers. Huh? Look, more lanterns. Sure, but what? It's a mob gathering. Come on, we got a fight on our hands to save Luke Ranger from being lynched. Now returning to Hopalong, Cassidy, and his story, the cold country. The site of a lynch mob gathering on the streets of Lode Hill has sent Hopi and California speeding to the jail, where Luke Ranger awaits his trial as the killer of his wife. I wasn't gonna let you in at first, Cassidy, then I recognized your voice. You must know about that mob then. Yes, I've known about it for the last hour. All I can do is wait for him to get here. This is the time when being a peace officer grates a man's hair. I don't envy a marshal. What about deputies? I had two of them here. The other one has a broken leg. Here they come. How's Luke taking it? Oh, he's worried about his boy more than anything else. I think he's innocent, Marshal. We've got to save him. Ever seen a lynch mob in action before, Cassidy? A few times. Were they stopped? Sometimes. Well, I've never seen one stopped. This time I got the job at trial. They're gonna start batting at that door in a minute. That's right. By the time they break in here, there'll be no stopping them. Somebody's got to go out there and talk to them. It won't work, Cassidy, but now they're all out of their heads. It's a lust for killing. They're not up to the door yet. Pull it open a little. Wait a second. What for? I'm going out there. Luke Granger deserves a hearing, and I'm gonna see to it that he gets it. Well, Hoppy, no. Man, you'd be committing suicide. That's my problem. Come on, Marshal, open up. Hey, now wait a second. Pulling a gun in the law. You're going crazy, Hoppy. Luke's gonna get his chance. Take his keys, California. Open the door. Hold it, Marshal. Now the bars. But Cassidy! Save it for later, Marshal. Get the door shut. Look who's here, man. It's the Hoppillong Cassidy. What's the matter, Cassidy? Get cold feet in there. Now, if that Granger's feet are colder... What do you want to do, Cassidy? Make a speech? You seem to be doing all right, Morley. Morley always did have a gift again. All right, Cassidy. What do you want? I've been thinking that you men aren't here for any barn dance, not with Morley doing the calling. We're here to hang Luke Granger for the murder of his wife. That's right. And if any of his friends object, we'll string them up too. So you're the voice of the people, are you, Morley? Never mind that. Just get out of the way. What's your stake in this, Morley? And don't say justice. From one of Bart Beleu's men, that'll just draw a laugh. He's got you there, Morley!  Thanks for reminding me of that, Morley. I'll put it away. I'm warning you, Cassidy. Get out of the picture before it's too late. You men, you're not all Beleu's hired hands, are you? He doesn't control that much of this town, does he? Beleu would like to see Luke Granger out of the way and you all know why. That's why Morley's here running the show. Is there one man here who actually saw Luke Granger shoot his wife? Cassidy, I'm giving you ten seconds. Ten seconds to ease out of here.  How about it, men? You'd hate to hang Granger and then find out later he was innocent, wouldn't you? Think you'd ever be able to get a good night's sleep?  How about it? Maybe this fella's right. That's all for you, Cassidy. I'm shutting your mouth as of now. I wish you wouldn't try him, Morley. This is where your string runs. What is he doing? Beleu. Somebody, go... Beleu. He's down. That fella beat him to the drum. Sorry, Morley. This time it was your string. He's dead. Morley's dead. Yes, men, and don't you think that's enough killing? Let's wait this thing out. Let's give Luke Granger a fair trial. Well, Cassidy, you did it. I wouldn't have thought it possible. The mob's all broken up, Poppy. Most of them gone home. Luke's like Luke's gonna be safe. For a while anyway, unless Beleu starts something new. I don't think Beleu will try anything else. You have a hard time arousing another crud. Just the same. We're gonna do any lasting good for Luke. We'd better be finding out something about his wife's death. Where was she found, Marshall? Right outside their home. Know who found her? I did. You did? I'm afraid I don't know you, or do I? Well, this is Joey Edmonds, Cassidy, one of Luke Granger's best friends. Glad to make friends with you, Cassidy. Thanks, and I'm glad to meet you. Luke's wife was shot, wasn't she? That's right. Anyone find the bullet? Don't believe anyone thought to look. I guess that's the way of it. Mind if I take a look, Marshall? You could make it legal by deputizing me. You practically deputized yourself, didn't you, Hoppy? All you need to make it complete is the badge. Talk about being cold. How long are we gonna stay here, Hoppy? All night? Move that lantern over this way a little. Must be two o'clock in the morning, and here we are out in the snow looking for spent bullets. Just one bullet, California. A little more with the lantern. I ain't never been so cold in all my life.  A little more. I got it up against the porch right now. Look, stuck in the post. That is a bullet, certainly is. Let me get it with this knife. There. Well, I'll be turned, but now you've got it. What are you gonna do with it? Take another look at it. It's a 38. Right. Now, how many fellas you know carry 38s? Not very many, maybe two or three. Which should narrow down our problem some. Come on, California, let's head for the hotel and some sleep. Morning, Miss McKenna. Morning, Mr. Cassidy. Nice having all the snow shoveled off the walks, isn't it? Yes. Here, let me help you with a few of those bundles. Thank you. Just a few things from the store. Wait, you don't have to take all of them? I kind of wanted to talk to you about Luke and some of Luke's friends. Well, you just come on inside. Here we are. You can set that bag of feed right here on the porch, if you will. I'll take it around back later. I'll carry it around for you right now. I'll sure be obliged. I'll go on through the house. I'll see you later, then. Miss McKenna. I'm all right. You sure? What happened? He stepped out of the parlor right after I closed the door, shooting the place to pieces. Who? Where? He's gone now. He went through that window at the end of the hall. Luke. It was Luke. There's the empty cell. Some of that mob must have worked on the window bars last evening while we were busy out front. Luke just kept quiet about it, then pushed his way out during the night. It just doesn't make sense. Why would he have given himself up in the first place? Don't look to me for the answer. Poor Luke must have gone plumb loco. You'd be the same way, Joey Edmonds, if you'd been through what he has. He must have come to the house because he wanted little Neddy. When he couldn't find him, he... Well, I can't blame him, that's all. Where was the boy? I was out to the store. I'd left him with Sarah Claiborne up the hill. Looks like Joey's right. Luke's gone loco. We got him cornered, Matt. Luke Granger. Where? Loft over Van Nair's forge. He's gonna make a fight of it.  Says we'll never take him alive. At times like these, I'd rather be punching cows again. Marshall, do me a favor. Hold off on Luke for a while. You mean talk to him? Think that'll do any good? Don't talk to him. Just don't do anything. It's just a hunch, but I've got something I'd like to work on. It'll only take a little while, and it might save Luke Granger's life. What are you doing? Well, I guess I've been doing a little breaking and entering, Miss McKenna. Had no idea you were at home. No. You thought I was with everyone else, didn't you? You thought, ah! That gun! Just found it in your dresser here. A .38 with three bullets fired. And it was a .38 that killed your sister. Are you sure of yourself, Miss McKenna? You shouldn't have kept it around. You're insane.  Partly because you hated her, but mostly because you wanted to get control of young Nettie. But Luke out of the way, that would have meant controlling the mine and the money. Drop that gun, Cassidy. So you're in on a two, Marshall. Makes quite a picture.  Don't move. This is a .45 I've got on. You're not a .38. Which means I'll have to use Miss McKenna's a shield.  No, but I can beat the life out of you. I'm losing all that killing. Grab my gun. Get the .38. Ah, sorry, Marshall. I just can't break. That's all, Miss McKenna. Let go of me. Sorry, the Marshall's all through, and so are you. To hop along Cassidy and the cold country. How do you feel, Luke? Not good. Gonna take me a long time before I feel good again. Maybe you ought to bring young Ned in and ride the Santa Fe with California and me. Might help you forget. I thought I had friends in this town. Thought I had friends. You do have friends, Luke. Plenty of them. With just a few bad ones for after you and moving too fast for other people to keep up with them. Maybe you're right, Happy. You always good taught me into seeing things your way. But it's a darn good thing you came here to pay me a visit. A darn good thing. Ah, either way, Happy, how would you ever get around to figuring that woman was behind all this? I know there was that bullet, but that wouldn't point to no female. I figured it was Miss McKenna because she tried to get away with a lie. Lie? Yeah. When she tried to make out that Luke had gone berserk, she said she saw him climbing out the window of her house. She claimed she closed the front door, and then he stepped into the hall shooting up the place.  You weren't in the house, was you? I didn't have to be in the house. She just couldn't have recognized Luke under those conditions, that's all. Can't prove it by me. She couldn't have seen him because she wears spectacles. She'd been outdoors where it was cold, and she stepped into a warm house. From the second she closed that door, she couldn't have recognized an elephant in that hall. And those specks of hers were still fogged up as she stood there telling me about it. We'll see you with us, won't you?