 I'd like to thank the organizers for inviting me to give a talk here today. This is way far. There we go. So like Richard said, I'm a postdoctoral researcher in the laboratory of Claire Halpin, where we're looking at using plants or improving plants for making biofuels. So why are we interested in using plants to improve biofuel production? Well, they can take out part of that. They use CO2 in photosynthesis to, by growing them and using them as fuel, we're partially mitigating the effects of burning that fuel. Now the problem currently is that most, actually currently most biofuels is being used are first generation biofuels. I think in fact, I have a statistic from 2012 which stated that almost 40% of the US maize crop is going into fuel production. Now this has obvious effects on food prices and things like that. So what we're looking at is moving this along into second generation biofuel production, which is using glucose locked up in the plant cell wall and using this to make our fuels. Now this has just started. I think they're now in limited initial production of making cellulosic ethanol. And the reason for this that is just getting started is that the process of secarification breaking down the cellulose into its glucose units is very difficult because cellulose is bound up and interconnected with other plant cell wall polymers including hemicellulose and what we're interested in lignin. So this is just on the right is a simplified diagram of the biochemical pathway to synthesize lignin from its precursor phenylalanine. I just want to point out a few things from this pathway. If you listen to the Clint Chappell's talk earlier, you'll know that lignin monomers come in three different flavors, H, G and S lignin. And also a few of the enzymes that I'll be dealing with CCR, CSC and C4H. Just to know that these are some enzymes that help catalyze the formation of these lignin monomers. So we're actually using a plant called Arabidopsis thaliana. Now we don't use this as an ultimate end product for biofuel production because it's fairly small. But it is a good genetic model system. It has great established genomic and genetic research tools. It has a sequenced and well annotated genome. And it also has a fairly rapid lifecycle and prolific reproduction that allows for easier study. With the hope that whatever we learn in Arabidopsis, we'll be able to transfer into useful biofuel crop plants. Now we're taking two... I'll talk about two strategies that we've used to try to make these plants more amenable for biofuel production. So first I'll talk about a little bit about what we've done with some co-expression analysis, which is basically looking at a plant under many different conditions, as many conditions as you can get, and looking at genes and which ones are expressed together. With the hope that by looking at the genes that are expressed together you should be able to find ones that have a common process. And the second is mutant screening, which is basically mutating a plant and looking for a specific trait that you're interested in. And in this case we're hoping for increased scarification. So I'll go back to the code. I'll just start off with co-expression analysis. So what we did was with the co-expression analysis, we looked at the expression of several different lignin genes. And I should mention this is in collaboration with our collaborators at the University of Ghent, Orian's lab, and at the University of Wisconsin, John Ralph's lab. So just go back to the point that we took... we looked at the expression of several different lignin biosynthetic genes under all these different conditions, and looked at what genes came out as being expressed together with these lignin biosynthetic genes, and one that popped out, along with several other lignin biosynthetic genes, one that popped out that wasn't really known to us was this gene that we termed CSE. Now, since our labs tend to look at lignin, the first thing we did was to check to see if the lignin levels were altered in a mutant of this CSE gene. And it turns out lignin content is reduced on the left graph here. You can see the wild type or our normal plant has a certain level of lignin, but our CSE mutant has a reduced level of lignin. And also, not only does it have reduced lignin content, its structure is altered. So a different type of lignin is being made using two different... two different assays of measuring the different lignin monomers. We see that in the CSE mutant, the H units represented by the red bar are increased over the wild type. So that suggests to us that not only is the lignin content reduced, but its structure has been altered as well. So altering the lignin is nice because we studied lignin, but it also would be more interesting to us, and for the GSEP project, is if secarification was affected since greater secarification equals more sugar, which equals more biofuel. So when we tested the mutant for secarification, we find that, yes, its secarification is increased. The mutant, the CSE mutant here is represented by the red line, and you can see that compared to the black lign, which is the wild type or the background plant, or the normal plant, that we're getting almost four times as much sugar under these conditions than the normal wild type plant. So it suggests to us that the CSE gene could be a target for improving crop plants for biofuel production. And we're actually currently working towards applying this towards crop plants. So by basically studying the gene sequences, we have identified a copy of the CSE gene in several crop plants, as rice, it's present in switchgrass and poplar as well. And just as an initial indication of whether this gene would have the same effect in the crop plants as it would in Arabidopsis, we did a simple experiment where we took a regular rice CSE gene and transformed it into our Arabidopsis CSE mutant to see if that would rescue it. And when we do that, you can see this is just a simple growth assay. Since our CSE mutant has a slight growth defect, when we transform it with the rice copy, we get restoration of the wild type growth. So that indicates to us that the rice CSE gene can perform the same function as the Arabidopsis gene, which would then indicate that if we did the same thing in rice, we might be able to get the same effects. So I'll just transition quickly into the other method that I was speaking of, which is mutagenic screens. So I've actually performed two mutagenic screens. The first one I'll talk to you is enhancer screen. So we took a lignin biosynthetic mutant, turn breath 3-3. You listen to Clint Chappell's talk, you should know that this is a mutant in one of the lignin biosynthetic genes. Now it's not a severe mutant. It has virtually no growth defects, but it does have a slight decrease in lignin and a slight increase in scarification. But what we're looking for is an even greater increase in scarification. So we mutagenize these plants and by using a high throughput scarification screen developed by collaborators at University of York, we look for plants that basically give us more sugar than the background ref 3-3 mutant. And when we identify those mutants, we can go back to the progeny and hopefully characterize it further. So this is just a graph of our scarification. WT is wild type or normal plant. And then the background mutant, the ref 3-3 mutant, you can see has improved scarification. But we also identified several mutants, actually most turned out to be alleles or basically versions of the same gene that's been mutated that have an even greater increase in scarification. In fact, you can see here that we're almost getting 100% conversion of the cellulose in these plants in the glucose. Now, this is not necessarily a trivial thing because anecdotally, when you cross two separate scarification improving mutants, you don't necessarily get an additive increase in the scarification itself. So this seems to indicate to us that this mutant could be especially important in improving the scarification of plants. Now, like I said, we're a lignin lab, so we're interested in the lignin content of these plants, and we asked the question on whether this improvement in scarification was also due to reduction in lignin. So we did some lignin assays, and in the graph on the top is just a graph of the lignin content of these plants. You can see that in our mutants, our lignin content is reduced even more over the background mutant. So also when we do staining for lignin in some stem sections from these plants, that the background mutant still has some lignin content, as you can see by the pink color, but our double mutant has an even greater decrease in the lignin. So that seems to indicate to us that our scarification increases are due to lignin decreases. Currently, we're working on trying to altering these genes in some crop plants to see if we can get the same effects. Just quickly, I'll go on to our second screen, which is a similar process, but a different question. So in this case, we're also looking at a background mutant in the top left plant there. Another lignin mutant has great increases in scarification, but it's also dwarfed because of this mutation. Now that doesn't really help us, because if we want more sugar out of these plants, reducing the size of the plants isn't helping us. So we mutated these plants and just did a simple visual screen looking for plants that have rescued size. Again, it's the case of identifying these mutants, going back to their seeds, and hopefully identifying those mutants even further. Now when we screen these plants, we were hoping to find several different mutants, and what was interesting is that we found basically two different types of plants. One is a lot of mutants are like the ones on the left here, where the size is fully rescued back to the wild type size. But we also found a few mutants like those on the right here, where it's intermediate in between our mutant and the original wild type plant. So now it wouldn't be helpful if we just reverted the size back to our wild type, but we lost the gains in the scarification. So we performed scarification on those mutants, and you can see here, this is our wild type plant. Our background, our background CCR1-3 mutant, this lignin mutant that we use for the screen has increased scarification. And then just as an example, several mutants still maintain that increase in scarification. Similarly, let me do this quickly, these mutants also maintain their reductions in lignin content, which seems to indicate that us, converting the phenotype of the plant back to the wild type, or changing something else, that will improve the plant for the... will improve the plant that makes it better so we can get more sugar out of these plants. So quickly, I'm over time already. Just as a summary, so using co-expression analysis, we were able to identify CSE as a gene as target for improving plants for biofuel production because by altering this, we were able to get more sugars out of these plants. And we're also looking at other mutations to hopefully either... that we can rescue growth also with greater improvements in scarification, with the idea of being by generating all these different mutations that we could hopefully combine them to produce plants that have the properties that we want, increase the scarification without harming the plant, without having a size penalty that really is too deleterious for use for this biofuel production. So I'd like to acknowledge GSEP for their funding. Everybody in our lab that helped with this project, also our collaborators, Boryan's lab and John Ralph's lab at the University of Wisconsin collaborate with us on the CSE work. Simon McQueen Mason's lab at the University of York really helped us out with the high-throughput scarification screen. And we'd also like to acknowledge Clint Chapel's lab and Sirius Lee's lab, who are co-collaborators on a new GSEP grant. So thank you, I'll take questions. So what do you use for the mutagenesis in these? We use EMS, ethyl methane sulfonate. And what sort of fraction of the mutations turn out to be something interesting? Well, fractions of plants is very low. So you have to screen thousands and thousands of thousands of plants to find one that you're interested in. That's why these high-throughput screening techniques are so useful. Fractions of mutations is quite low as well. Usually, even though we're screening thousands and thousands of plants, each plant that we're screening might have 20 to 30 useful mutations throughout the genome, but they're hitting things that we're not interested in. So it's quite large numbers. Thank you.