I'd like to thank the organizers for inviting me to give a talk here today.
Oo, this is way far.
There we go.
So, like Richard said,
I'm a postdoctoral researcher in the laboratory at 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 the, they can,
they use CO2 in photosynthesis, so 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 as being used are first generation biofuels.
I think, in fact the statistic from 2012, which stated that almo,
almost 40% of U.S. 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 there are now in limited initial
production of making cellulosic ethanol, and the reason for this,
that it's just getting started is that the process of saccharification,
breaking down this cell, breaking down the cellulose into its glucose units is
very difficult because cellulose is bound up and interconnected with other plants,
cell wall polymers including hemo, 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 Chapple's talk earlier you'll,
you'll now that lignin monomers come in three different flavors.
H, G, and S lignin and also the, a few of the enzymes that
I'll be dealing with CCR CSE,
and C4H, just to know that they're, these are some enzymes that help it catalysi,
catalyze the formation of these lignin monomers.
So, we're actually using a plant called Arabidopsis thaliana.
Now, we, we don't use this as a ultimate end and 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's, has a sequence and well-annotated genome and it also has a ver,
fairly rapid lifecycle and a shu, and prolific reproduction that allows for
easier study with the hope that whatever we learn in our 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 the genes, and
which ones are expressed together, with the hope that by looking at the genes
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 that you're interested in, and
in this case we're look, hoping for increased saccharification.
So I'll go back to the co, I'll just start off with co-expression analysis.
So what we did was with the co-expression analysis we took,
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, or, Orion's lab at the University of Wisconsin, John Ralph's lab.
So I just go back to the point that we took,
we looked at the expression of several different lig,
lignin biosynthetic genes under all these different conditions, and looked at what
genes came out as being expressed together with these lyg, 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 turned CSE.
Now CS, since our labs tend to look at lignin,
the first thing we did was to check to see if the lignin levels were,
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 mea,
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 in the wild type, 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 study lignin but it also,
it would be more interesting to us and for the GCEP project is if
saccharification was affected, since saccharification,
greater saccharification equals more sugar, which equals more biofuel.
So when we tested the mutant force saccharification
we find that yes that's its saccharification is increased.
So the mutant, the CSE mutant here is represented by the red line, and
you can see that compared to the black line, which is the wild type or
the background plant or the normal plant that we get, we're getting almost four
times as much sugar under these conditions than the normal wild type plant.
So it, 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 using, by basically studying the gene sequences or identif,
if we have identified a copy of the CSE gene in several crop plants such as rice
and there's sweet, its presence which grass 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,
just 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 could 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 in wild-type growth, so
that indicates to us that the rice CSE gene has, can perform the same
function as your 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 muta, mutagenic screens.
So I've actually performed two mutagenic screens.
The first one I'll talk to you is an enhancer screen.
So we took a lignin biosynth, biosynthetic mutant termed ref3-3
you've listen to Clint Chapple's lab or talk, you should know that this,
this is a mutant in one of the ligament biosynthetic genes.
Now it's not a severe mutant, it has virtually no growth defects, but
it does have a slight decrease in lignin as slight increase in saccharification,
but what we're looking for is an even in, greater increase in saccharification.
So we mutagenize these plants and by using high-throughput saccharification
screen developed by collaborators at University of York we look for
plants that basically give us more sugar than the background ref3-3 mutants, and
what, when we've identified those mutants we can go back to the the progeny and
hopefully characterize it further.
So this is a mea, this is just a graph of saccharification.
WT is wild type, or normal plant and then the background, a mutant.
The ref 3-3 mutant you can see has improves saccharification but
we also identified several mutants.
Actually, most turned out to be alleles or ba, basically versions of the same gene
that's been mutated that have an even greater increase in saccharification.
In fact, you can see here that we're almost getting 100%
conversion of the cellulose in these plants, into glucose.
Now, this is not necessarily a trivial thing because anecdotally
when you cross two separate saccharification improving mutants you
don't necessarily get an additive increase in the sacchar, saccharification itself.
So this seems to indicate to us that this mutant could be
especially important in improving saccharification of plants.
Now like I said, we're in lignin labs, so we're interested in the lignin
content of these plants, and we ask the question on whether this improvement,
this saccharification was also due to reduction in lignin.
So, we did some lignin assays, and on the, in the graph on the top
is just a graph of the lignin content of these plants, and 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 ref, or
background mutant still has some lignin content, as you can see by the pink color,
but our double mutant has an even greater increase in the, or
even de, greater decrease in the lignin.
So that seems to indicate to us that our saccharification increases are due
to lignin decreases.
Currently what we're working on trying to get these gene or,
altering these genes in some crop plants to see if we can get the same effects.
Just quickly I'll go onto our second screen, which is a different,
it's similar process, but a different question.
So in this case we're also looking at a background mutant.
In the top left plant there, we have a plant,
another lignin mutant has great increases in saccharification, 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'd we mutated these plants and just did a simple visual screen,
looking for plants that have rescued size.
A si, again, it's a simple, it's the case of identifying those 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, or one, a lot of mutants are like the ones on the left here,
where the size is rescued back, fully rescued back to the wild type size,
but we also found this, 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 those back, if we reverted
the size back to our wild type but we lost the gains in the, in the saccharification,
so we perform saccharification on these mutants and you can see here,
this is our wild-type plant our background, our background ccr1-3 mutant,
this lignin mutant that we used for the screen has increased saccharification.
And then, just as an example, several mutants
still maintain that increase in saccharification.
Similarly, do this quickly these mutants also maintain their
reductions in lignin content, which seems to indicate to us,
we're not just reverting the phenotype of the plant back to the wild type,
we're changing something else that will improve this,
will improve the plant for the will improve the plant that makes a variant so
we can get more sugar out of these plants.
So quickly, I'm overtime already just as summary.
So using co-expression analysis,
we were able to identify CSE as a gene that's target for
improving plants for biofuel production because by altering this
we're 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 and saccharification,
with the idea being by generating all these different mutations, that we could
hopefully combine them to produce plants that have the properties that we want,
increase saccharification without harming the plant, without having a size penalty.
That really is too deleterious for use for these bio, this biofuel production.
So I'd like to acknowledge GCEP for their funding everybody
in our lab that helped with this project, also our collaborators, Wout,
Wout Boerjan's lab and John Ralph's lab at the University of Wisconsin collaborated
with us on the CSE work Simon McQueen-Mason's lab at the University of
York really helped us out with the high-throughput saccharification screen.
And we also like to acknowledge Clint Chapple's lab and
Serious Li's lab who are pro-collaborators on a new GCEP grant.
So thank you, and I'll take questions.
[APPLAUSE].
>> One at a time, has anyone has one question?
>> So what you use for the mutagenesis in these?
>> We use EMS, ethane methyls, eth, ethyl methanesulfonate.
>> And, and sort of a,
what sort of fraction of the mutations turn out to be something interesting?
>> Well fractions of plants is very low, so I mean you have to screen thousands and
thousands and 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.
[LAUGH].
>> Okay, thank you Chris.
[INAUDIBLE].
>> Thank you.
>> [APPLAUSE]