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Mechanisms of chemical diversification in plants

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Mechanisms of chemical diversification in plants
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biosynthesis drug Squalene metabolic plant sterile chemical Terpenes molecule enzymes color Common Aroma compound Bioverfahrenstechnik membrane type areas precursor synthetic biology Polymerdispersion synthase chemical conditions metabolic pathway function Gamma Terpenes Steroids Sterine
Squalene activities properties precursor scaffolds potential Säuren DNA man Oleanolsäure Terpenes molecule samples Magnetometer protein function cell chemical structures Beta derivatives doses
physical chemist sugar chain type growth operation Süßholzwurzel set gene cluster chemical cancer molecule Terpenes enzymes Horizontaler Gentransfer modifications cyclase lesions Medroxyprogesterone Metabolit properties scaffolds steps gene Flint genome complexes firm toxicity van clusterization model blue oxygen resistance amplifier sense biosynthesis bioactive species Cross section (geometry) Squalene Stimulant pathogens plant methyl man flow Magnetometer sweet tasting cell soil Mutagen Redoxreaktion Beta electron transfer mixture processes areas type acyl Trisaccharide P450 biosynthesis Barley wine fluorescence Anthranilic acid hybrid mutagenesis mutation glycosides conditions metabolic pathway Seedling function UV rays chemical structures mRNA extract base pair
acyl acyl species Bohr des steps scaffolds fluorescence plant gene complexes man Noma (disease) Vakuole molecule Terpenes conditions metabolic pathway yield protein plant soil electron transfer
areas organizations Chromatin plant gene gene cluster man clusterization coordination Gamma overexpression cell regulation report level
sugar synthase gene set cluster traces Terpenes molecule enzymes variants protein modifications transformations Medroxyprogesterone properties scaffolds steps Cyclooctatetraen firm systems rapid van GFP protein Gamma RNA species sequence biosynthesis species Squalene plant cytochrome case man sweet tasting plant overexpression report tool Beta glycosylation level processes mixture P450 fluorescence multiple Rohtabak Gletscherzunge sugar synthase agricultural initial metabolic pathway function chemical structures Terpene overexpression AdoMet
Index Glucose Sauce metabolic operation Anthocyanin gene cluster chemical Alfred molecule Terpenes chromosome enzymes Fructosebisphosphat <D-Fructose-1,6-bisphosphat> modifications cyclase carotenoids biochemistry segregation steps gene Gefäßverengendes Mittel genome Exon terminal Inositolphosphat-Synthase <myo-Inositol-1-phosphat-Synthase> clusterization match model Cycloalkane compounds oxygen amplifier biosynthesis Gum arabic species Squalene plant cytochrome Opium man originators clusterization Cadmium sulfide Magnetometer cell plant overexpression Common Beta level type natural products biosynthesis operation Arginine precursor Stickstofffixierung DNA Intron cluster glycosides level metabolic pathway Anthocyanin function Silencer (DNA) Peroxyacetylnitrat Medicine Terpene yeast Sterine Mars
plant Chromatin gene cluster DNA man cell overexpression Insulin Chrome level probe organizations Chromatin DNA fluorescence genome hybrid cluster clusterization metabolic pathway regulation AdoMet overexpression condensation
Gum arabic species Chromatin activities plant Histone gene genome cluster complexes cluster man Trihalomethane clusterization clusterization rapid metabolic pathway protein plant Genregulation overexpression modifications tool yeast
Glucose Gum arabic chain activities Bohr growth pathogens plant gene cluster case chemical heredity molecule Terpenes machine variants cell transcription overexpression level Einschlüssen Trisaccharide survive steps Histone gene genome firm mutation cluster toxicity Combining clusterization metabolic pathway compounds cell Mars overexpression
sugar metabolic plant gene cluster case heredity man Terpenes analogue enzymes Soda cell overexpression Methyltransferase <S> Common Beta level Angiotensin-converting enzyme type Metabolit P450 precursor steps assemblies gene genome firm cluster toxicity systems biological systems clusterization metabolic pathway model Seedling function Gasoline enzymes Terpene
Sauce sense Bohr plant RNA synthase gene man specific Terpenes stress cell tolerance plant Beta level Growth hormone type nitrogen steps synthase toxicity clusterization metabolic pathway function Primordial nuclide Silencer (DNA) Terpenes cell Zigarre stuff
Bohr serum metabolic gene cluster chemical genome alkaloid Terpenes blocks Terpene align chemical element Salami organizations Maische scaffolds gene genome source Glycin firm Mühle clusterization coupling Gamma amplifier Gum arabic biosynthesis species Transposon Rizinusöl plant cytochrome case man synthetic Zearalenone Magnetometer overexpression Inneren Rhizosphere type Synteny P450 Sparkling wine glycol balance multiple synthase glycosides chromosome Combining synthetic metabolic pathway function Terpene Steroids Mars base pair
right so now for something a little bit different I'm going to talk to you about what appears to be a horrific ly complex mess which I know isn't good for synthetic biology but I want to give you some reassurance that I think we can start to make sense out of all of this mess so as I'm sure many of you know plants make a huge array of different chemicals and these are the chemicals that protect plants against pests and diseases they attract herbivores and pollinator seed dispersal agents and of course we use them as flavors fragrances colors sense drugs for industrial biotechnology applications so there are hugely valuable resource but they're very very complicated and my interest in this area started with a particular group of chemicals called triterpenes which are one of the largest and most structurally diverse types of specialized in Taba light produced by plants and the triterpenes share a common pathway with sterile biosynthesis which of course is essential because sterols are essential components of membranes and they're also hormones so steroids are essential they're part of primary metabolism and sterols are made via the mevalonate pathway here which goes to this precursor to three oxido squalene and 23 oxido squalene is linear but it can be cyclized by sterile synthases implants this would be cyclo Artan all synthase to make the first committed precursor in the sterile pathway here cyclo Artan all which can then go on and be modified and turned into various ste roles alternatively this molecule can be cyclized 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 pros us 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 oxido squalene and i'm going to focus in a little while on this one here which is called beta amarin it's the Penta cyclic triterpene it's one of the most common triterpenes found in plants so beta a marine happens to be very common in plants but it's also a precursor of
sorry before I say that I've just mentioned that these are some of the alternative cyclisation products of 23 oxido 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 and they their structures are consistent with their being made by cyclisation of 23 oxido squalene so just with this very simple precursor we're already getting a huge amount of structural diversity and simple triterpenes and have important properties in their own right for example oleanolic acid which is the derivative of beta amarin 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 in the therapeutics but of course the molecule like this is pretty difficult to modify chemically selectively because the scaffold has very little functionalization to work with so simple triterpenes are important but implants 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 amarin 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 oxygenation as they have sugars here is an acyl 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 triterpene so that we can do systematic analysis to understand the relationship between structure and function and you can see the property is listed here a diverse this one which I'm going to come back to happens to fluoresce bright blue because it has an N methyl anthranilate 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 genes-- avena and it's antifungal it also is phytotoxic so it's a natural herbicide and then you can see down here we have another try to have been produced by P called chromis happen in one and this instead of being a fighter toxic 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 anti-freedom properties whereas this one from licorice is 50 times sweeter than sugar and then we have various pharmaceutical properties listed here as well so these are just 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 a Venus in a 1 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 turn at the University of Oxford who had seen that the tips of oat roots avena fluoresce 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 Vita a marine scaffold the fluorescent and methyl anthranilate group here and a trisaccharide chain so this molecule is produced specifically by oat so most of these specialized chemicals produced by plants are only produced by particular narrow taxonomic windows so they are that's why they're cool specialized 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 Oates 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 out and we treated it with a chemical mutagen and we simply screened by putting germinating oak 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 triterpene biosynthesis for any plant species oats the very unusual amongst the cereals and grasses in making antimicrobial try to whereas the dicotyledonous plants make a huge array of triterpenes so this was an unusual situation in the grasses when people were interested in why oats were making this and other cereals such as wheat and barley in maize were not so we did this mutagenesis we got a bunch of mutants in fact we have nearly a hundred meetings 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 out and this has been challenged with the soil borne pathogen that causes massive disease losses on wheat and it's resistant but 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 which is involved in protection against soil borne pathogens and we would have stopped there because diploid out is not a model species by any means it's not a rabid opsys and this is a very very complicated molecule but the reason why we kept going was because the genetic started to tell us that something very strange was happening the low side that we defined by mutation but had not yet characterized were not Co segregating when we did genetic analysis they were sticking together and this suggested that the genes for this very complex pathway were physical ethically linked in the genome and many years on we've now characterized all of the genes in the pathway here i'm just showing 5 this is the gym for the first step the second step these are three genes for the acyl 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 we find and I should emphasize that these genes all in code highly different types of enzymes this is the occido squalene cyclase that converts 23 oxido squalene to beta amarin this is a p450 that introduces modifications to be to a marine this is an acyl transfer oz of sugar transfer as 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 the general understanding was that genes for the synthesis of specialized 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 defense the molecule if you take a cross-section of a note root is in the epidermis which 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 hybridization 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 coexpression 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 characterized all of the genes and ends on now we've just 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 transfer 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 and 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 an timo antimicrobial triterpenes 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 coordinate 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 11 partial explanation could be that physical clustering enables a higher level of regulation of the GNex of gene expression at the level of chromatin and potentially also higher nuclear organization 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 rabid opsys and rice and legumes this is just with the promoter for the first gene in the oak cluster but it's true also for the others we see Gus expression in the root tips and the lateral root initials that aim in these diverse plant species so remember that this pathway has evolved recently it's specific to out and so this provides a further conundrum it looks as though that the promoters for the Oak 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 23 oxido squalene could be cyclized it can be converted into all of these different cyclisation products by enzymes known as 23 oxido squalene sigh closes and i showed you examples of all of these diverse scaffolds and we now know whoops there in many years on we now know a lot more about the enzymes that make these scaffolds we still haven't characterized very many of them but these are some examples the oat one is up here interestingly dicots make be to a marine 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 and the tends to be most of these enzymes are specialized some of them make multiple products and 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 scuffles in different places and these are all betta a marine modifying cytochromes p450 not just from oat but from a whole range of other plant species so we're putting together now collectively 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 be to a marine modifying p450s there are also people 50s available now that make other scaffolds which we and others have characterized and you can see that we're building up a very powerful set of 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 psych laces and then the p450s to get to the 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 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 in oat it is and but one of the things that's really accelerated what we have been doing is this very nice transient plant x system developed by my colleague George llamanos off 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 cowpea mosaic virus that if you have a little bit of cowpea mosaic virus sequence from the RNA to gene of cowpea mosaic 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 cowpea mosaic 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 Agri bacterium 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 benthamiana 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 the leaf expressing our beta a marine synthase and this is a pea could be to amarin so this is the black scaffold here and then here we have the second step in our own pathway which is a very interesting cytochrome p450 which we knew modified be to amarin but we weren't sure exactly how and when we co infiltrate the genes for these two constructs the beta amarin 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
we've now gone on take now triterpene toolkit you'll hear more from Jim about the common plant syntax and the ways in which we've been getting our DNA synthesized in formatted so that we can mix and match index change with others so we have a toolkit of genes and enzymes for triterpene synthesis modification to make known and novel molecules and we can now play with these we put them into the the Nakota yana 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 triterpenes 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 bioessays and recently somebody in the lab has now made a gram of beta amarin 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 gene cluster there was only one other example of a gene cluster for a specialized metabolic pathway in plants and that had been reported by alphonse gill in munich who was working on maze on molecules known as ben's oxygen nodes which are also implicated in plant defense 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 amazed 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 in genetics it's well known you could say well our answer Cylons really special because plants all plants may come to silence carotenoid genes similarly are dispersed carotenoids are also very widely distributed nevertheless there are examples of other specialized metabolic pathways for example for glucose in alerts in brassicas that are not clustered so we wanted to find out how common this clustering phenomenon might be there was the original Mays cluster the oak cluster so we went to a rabid opsys because at the time the word many plant genome sequences available and the arabidopsis juno was very high quality and we did a very crude mining experiment we simply looked for genes that were predicted to encode 23 oxido squalene sigh closes so genes that work that look like they might be able to take 23 oxido squalene and turn it into one of those cyclic products that I've mentioned and in the arabidopsis genome there are 13 genes that belong to that family one of them is required for the synthesis of cyclo arsenal 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 sterile biosynthesis this is one of those jeans 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 but have not arisen by tandem duplication they're very different and in a Cell 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 benfield 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 over expression lines and silence lines and biochemistry and he was able to show that these genes did indeed form a pathway and this and was we called it rather mischievously the first example of an operon like G cluster in arabidopsis it's not an operon as I say but the point is these genes are nottttt under duplicates they encode steps in a pathway they're co-expressed so they have operon light 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 that the first one was for the synthesis of modification of Sally arnel which is a triterpene and then another one for the synthesis and modification of Mar neural another triterpene i should say at this point that although the oat avena sin pathway and the arab adopts this 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 more neural 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 mean we it's possible there was an ancestral gene pair that was the foundation for both of these clusters but we can't be sure of that and this contained knocks a desk waiting 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 that leave these clusters may form now since the maze and the oat story and the arab adopts this work others were discovering other clusters in other plant species work from Reuben Peters lab and professor Yemenis lab in in Tokyo hubs led to discovery of clusters in Ryan again for the production of defense-related compounds diterpenes 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 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 maze compound a rabid opsys there have been clusters reported for the synthesis of anti-nutritional compounds in tomato medicinal drugs in poppy these are the rice diterpene clusters interestingly the cyanogenic glycosides which have traditionally been thought of as the most ancient and highly conserved group of plant natural products and these are sporadically distributed across the plant kingdom and 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 organization 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 and 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 organization and I've shown you that 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 Sally Arnold cluster and you can see their expression as 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 hybridization with the oat 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 there 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 just descriptive but it suggests that Chrome's to chromatin d condensation 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 leave 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 and so in a rabid opsys for example genome-wide chromatin immunoprecipitation has enabled regions to be identified that are marked with a histone modification this is histone three k-27 trimethylation and this is a usually a repressive marking and these clusters have a very very discreet and localized marking of this h3k27 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 to a protein with histone h2a said which again has been associated with poising and with with with readiness of jeans 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 jeans with h3k27 trimethylation is well established implants 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 Sally Arnold pathway in arabidopsis and this is the histone 3k 27 trimethylation marking which is very clearly significant and pronounced and we can actually go through data from the Arab adopts asst genome we can look for windows of coexpression we can also look for windows of h3k27 trimethylation in tissues where the pathways are not active and we can use that to find new candidate pathways similarly i mentioned
that h2o zedge this alternative histone to variant is associated with pathway activation and we can use mutants to look for windows in the Arab docsis genome where we see a very clear effect on transcript levels of cluster components so this is the Sally onil cluster this is in the wild-type line this dotted line but in a meeting that is lacking h-2a said you can see that the transcript levels go right down and this is this very discreet windows is very striking similarly for the mar neural 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 will turn ugly Walter into a machine for mining plant genomes for discovering new pathways and chemistry's so the arguments for clustering I've talked about the 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 specialized 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 triterpene leads to a very sick root phenotype and here inside the wild-type this is normal wild-type route cell this is inside the mutant the epidermal cell layer is very distorted through these big membranous sacs and this is kalos training an accumulation of kallos 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 route phenotype although clearly we don't make the molecule so this is a toxic intermediate effect similarly in arabidopsis if we mess with the Sally Arnold cluster or the Mar neural 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 active molecule you can also generate molecules that are very bad that caused some 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 the population genetics in the audience geneticists in the audience was another level obviously so care expression Co inheritance 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 on 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 a certainly in this 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 transferases a cell transposes methyltransferases 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 stearyl pathway but then it has diverged it has acquired a new function and also a different expression pattern somehow then this gene has seeded the formation very 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 know them and so I've shown you examples of toxicity when you accumulate intermediates there also some other very subtle effects and this is
one of them so going back to our oats this is the the wild-type outline this is a mutant that is blocked in the first step in the pathway it's not able to make the beater amarin that is the precursor for the rest of the pathway at least not in significant quantity this is a late pathway enzyme this is the ace I'll transferase these roots all look normal and we noticed and were growing these seedlings on big square petrol dishes and we had to do that to see this effect because initially we were growing them on small pet read issues we kept thinking the roots of this mutant looked shorter that 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 amarin than the wild-type so this is a bit of a marine mutant that is blocked in the first step in the pathway there's still a basal level is a very low level of beta a marine detectable in the wild-type and a trace detectable here but these mutants are accumulating 40-fold more beta a maroon which as I said is a common plant metabolites and when we look closely at
what's going on in these roots is that 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 amarin 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 amarin 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 emerged 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 am room in the mutant we detect as I say a lot more and what we're seeing is actually a change in self 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 they're 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 psycho lawton all synthase gene you duplicate it it acquires a new function the ability to make beta amarin 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 Sally Arnold pathway in arabidopsis but we also see some more subtle effects where if we accumulate elevated levels of thaliana Lin the roots we actually see longer routes if you want to make longer routes to make a fitter 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 luteal and there is a loopy all synthase which is expressed in nodules when we silence it we see a window of clearly elevated nodule formation so loopy all appears to be suppressing nodule formation so we don't understand all of this but I'm just saying this stuff going on and it gets more complicated
and so these are just more examples of different effects that we can see when we tinker with clusters faster 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 and the whole clustering phenomenon and one of the things that we've been doing this is with Alex luton of 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 I won't go into but it's it is a massive family of diversity in plants and what Alex did was he mind these genomes for all terpene synthases genes so these are the engines of them generating diverse scaffold and he also mind them for all cytochrome p450 genes and then he asked using a sliding window approach how often he found a terpene synthases in proximity to a p450 gene relative to what would be expected by chance and the answer was very often and so we're able we've classified all of the terpene synthases 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 synthases 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 synthases genes that have already been reported I've told you about the diterpene clusters from rice he also found steroidal glycol alkaloid cluster from tomato the arab adopts these 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 evolved was he took a couple of the major pairings the major groupings of terpene synthases mmm and p450s and this is a little bit just bear with me this is some easy to understand once you 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 synthases within a pair and asked which one was the most closely related terpene synthases in the other pairs and then he did the same for the p450 partner of that terpene synthases and what he found was something very interesting which was that in the dicots the terpene synthases 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 synthases 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 synteny in the dicots and this is kind of the opposite way around the way that it's normally thought of I've spoken to a lot of genomics experts and they don't know how to explain this and we're only looking at this on the basis of terpene synthases not the whole organization of the genome but it suggests that there is micro syntenic duplication in the dicots and that would be consistent with our Arab adopts this hypothesis for them Sally onil and Mar neural clusters but in the monocots it's all chopping and changing and de novo combinations he also looks at all of the genes within these pairs and compared them to genes across the genome looked at the frequency of transpose vil elements in the flanking regions because you might expect that trans poseable elements would play a role in moving jeans around and as we might expect he found that they were sick frequently more transpose ax Bilel iment in the flanking regions of the genes within these pairs than in the ones that were not prepared 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 diterpene synthesis in castor this was another cluster in arabidopsis again to try to fumes 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 chemistry's so in summary I just like to say that i've talked about examples of these metabolic gene clusters in plants with the synthesis of diverse chemicals we don't yet know what the balance will be of pathways wear the jeans are dispersed relative to those that are clustered nevertheless clustering is very useful for mining genomes to discover new pathways and chemistry's we can look at these clusters to try to understand what they're 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 oak 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 arabidopsis roots and this opens up opportunities to start to tailor the rhizosphere by making designer chemicals implant roots and with that I'd like to finish and just draw your attention to the many people and who have contributed to this work and also the funding sources and I think you'll hear more about open plant from gym in the next talk thank