My name is Philip Benfey I'm a professor in the Biology Department at Duke University. I'm also
an investigator for the Howard Hughes Medical Institute. Today i'd like to tell you about some
of the work that we do with root genetics. And you the first question you might ask is why even
study plants, much less roots? And I would present to you the idea that plants are actually at the
center of some of the major problems facing the world today. First one would be climate change.
Climate change is affecting both agriculture and, in all sorts of other ways, affecting the
way plants grow and survive. But I also present to you the fact that plants can have a positive role
in mitigating the effects of climate change. Secondly would be nutrition and health.
That is, plants are the source of much of our diet and there are many malnourished people in
the world or undernourished and plants play a very important role in combating malnutrition.
Lastly I would say for clean energy. There are lots of uses of energy that need liquid forms,
transportation fuels, jet fuels for example, and plants are an alternative for providing
a liquid form of clean energy for those uses. So coming back to roots, we study roots for many
reasons that i'll tell you about but most people don't think about roots because you don't see
them. They're out of sight out of mind. But they also, for the plant, play a really critical role.
They're the major place where plants acquire their water, water comes up through the roots. Also
nutrients, nitrogen phosphate etc, is acquired by the plants from the roots. Roots also provide
anchoring, without roots plants fall over. And finally, roots are the place that plants interact
with microbes in the soil, including microbes that are, for example, nodules on soybeans that that
fix nitrogen. Now plants are not the same as animals in the way they grow. In an animal
you grow with the the embryo has almost all of the organs that you will have in the adult. And
when you go from the embryo to the adult mainly what you're doing is increasing the size of those
organs sometimes increasing their complexity. For a plant in the embryo you really just have a
mere sort of outline of what that plant will look like. And the reason is that plants grow
from the top and the bottom with use of stem cells and from those stem cells you get every other part
of the plant. That is the the flowers the leaves and all the root tissue. As you look at the root
tissue, particularly the root tissue of the plant that we work on which is arabidopsis thaliana,
you see also some simplifying aspects, some of the things that attracted us to
work on roots in the first place. If you slice through the root you see that in
that circular piece of root each of the different colors is a different tissue within the root. From
the outside in that red tissue is the epidermis, then you have the blue which is the cortex,
inside that the green is the endodermis. And if you rotate the root
you'll find that you have exactly the same tissues in exactly the same places.
There is radial symmetry to the root and that greatly simplifies how one analyzes the organ.
Another great simplifying factor of the root is that the stem cells the cells that give rise to
all the other cells in the root are located at the tip of the root. And they are for arabidopsis at
least, that that part of the root is translucent, you can see right through it it with a microscope.
Now what i'm going to tell you about is how we've used genetics to analyze root
development. Genetics were actually invented discovered by Gregor Mendel using plants. In
this garden he looked at pea plants and saw plants that had different flowers different leaves etc.
Now when I started my independent career I looked for mutants in roots that were different from
the wild type, the normal. As you see here on the left are the that's the wild type root and
then we have two sets of mutants next to it. And the way we grow our our roots are on a flat petri
dish and the petri dish is placed upright so the roots grow along the surface of the petri dish
and we can see all sorts of differences. In this case it was a very simple difference.
The difference was just a matter of length of the root, one was growing a lot slower
than the other. And we named one of these mutants, perhaps not very imaginatively, short root because
it had a shorter root. And then only later we discovered another mutant. And then we realized
there was something more going on and we gave it a little better name which we called scarecrow.
Now what is going wrong with these roots? Why are they shorter? Well it turns out they're
actually missing one of those tissues that I discussed a minute ago. That is,
if you look at the wild type now we have a real section through the root on the left hand side
we have the outer layer of epidermis, the next layer in that is has the little black stars in
it is cortex, the layer inside that is endodermis, and then internal to that is the vascular tissue,
the very small cells. So there are two layers, each with eight cells. You look at short root,
short root has an outer layer of epidermis, it has those small cells in the middle the vascular
tissue, but in between there's only a single layer not the two layers that you see in wild type.
Scarecrow similarly, only a single layer between that outer layer and the central layer. And that
is sort of like, these are real tissues these are functioning tissues that play key roles. So
it's as if these plants are missing something like a kidney or liver in an animal. Now what
we do in genetics is try to understand what's really gone wrong with these. When we compare
the mutant to the wild type there's a great leap of faith of genetics, which is if we can precisely
define the difference between the mutant and the wild type, the mutant and the normal,
that will tell us what the gene does in the normal situation. Let me walk you through this. So when
we looked at the at short root and scarecrow we said why does it have only a single layer?
Well we realized that what happens at the tip of the root, there is a stem cell the green cell,
that divides first along the transverse axis. And it does that it has to regenerate itself. That's
what all stem cells do. The upper cell then, the daughter cell, divides along the longitudinal axis
to give the first two cells of the cortex and endodermis lineages.
The yellow on the outside is the cortex the blue on the inside is the endodermis. And so because
there's only a single layer in short root and scarecrow, that means that second division hasn't
occurred. So that's one of the things that both of these proteins must be doing in the wild type
situation. There's another thing though, as we looked at these mutants using different markers,
different ways of characterizing the the the actual cell types, we realize that in short root
it's actually entirely missing the markers, the characteristics of the inner layer of
the endodermis. So the division doesn't occur and it never specifies and endodermis. In scarecrow,
again, the division doesn't occur but we found characteristics of both endodermis and cortex
in that single cell layer. So what does this mean? It means that as we think about these short root
must do two things. The short root protein, that is the original non-mutated form, is critical
to get that division to occur and then specify the endodermis. While scarecrow, because it has
both endodermal and cortex attributes, its primary role must be to get that division to occur.
Once we had figured out what the mutants are probably about, that is what the wild type
does and what the mutant is missing, our next goal was to identify the actual gene that had
been disrupted in the mutant . We found scarecrow and the next thing after that,
we identify the gene, we clone it, that is we isolate the DNA, and then we said well where is
that gene normally expressed? And we can look at this in a number of ways. What i'm showing here is
where we use its promoter and we drive the green fluorescent protein, this is something that came
out of jellyfish that when you shine laser light on it it becomes green. So the green part here is
where this gene would be expressed if you used its promoter, the regulatory part of that gene,
fused to GFP, you see more or less what we would have expected. That is, it's expressed in that
stem cell prior to the division and is expressed in all of the endodermal cells after the division.
And the same is true for the protein. For the protein what we did was we used the same
regulatory region, we fused it to the coding region for scarecrow, that is the part of the
DNA that codes for the protein, fuse that directly to GFP so we have a fusion protein
of scarecrow with gfp, put that back into a mutant, show that it actually rescues the
mutant phenotype, it looks wild type now, and again it looks exactly what like what we would
expect. It looks very similar to the RNA. When we looked at short root however, we got a surprise.
Short root, its RNA expression either using the GFP approach which you see on the right, or using
a probe that we're looking directly at the RNA, this is called an in situ hybridization on the
left, it's not where we expect. It's in the small tissue cells of the vascular tissue in the center.
It's not in the cell that has to divide, it's not in the endodermis. So how could it be sending the
information over to get that division to occur and the endodermis specified? Well in animals,
it's well known that transcription factors, these are genes that make proteins that bind to DNA,
that these can go into the nucleus where the DNA is and turn on other genes, make small
proteins that would then move from one cell to the next. They would interact with another protein,
a receptor and then we get other things to happen. We have another hypothesis to test
which is in plants there are channels between adjacent cells, these are called plasmodesmata,
and proteins can actually physically move from one cell to the next. And this turned out to be what
was happening in short root. So on the left again we have the RNA expression short root promoter,
the regulatory region of the DNA fused to GFP so this is the what the RNA looks like.
On the right we see the same promoter driving short root coding region, fused to GFP and now
you can see that the protein is found exactly where we would predict. That is, it's found
in the stem cell prior to division and in all the endodermal cells. And it's found in the nuclei
where the DNA is in all those cells. So why have it expressed only in that location? What happens,
why not just express this protein, if it's so important, in just the the endodermal cells
and the stem cell? So we ask this question by using the scarecrow promoter now,
the one that's expressed only in the endodermis. If we drive the short root protein with the
scarecrow protein, what happens? What happens, as you see in the image on the right,
is we get something that looks like a cut off tree trunk. We get lots and lots of new layers.
So clearly there's something about the regulation of short root, having to only move one layer over,
that really is critical to getting the right formation of the tissues in the
right places. So in summary what i've told you about is that studying plants can help address
some of the major issues facing the planet today. And when studying plants a focus on roots
is important because roots are really critical for plant growth and health. Then I described
a genetic screen for mutants with shorter roots. And somewhat surprisingly from that
mutant screen we found two mutants where there was an entire cell layer that was missing. These
were short root and scarecrow and then we looked closer at short root and found that
its gene product a transcription factor actually physically moves from one cell to the next.
Expression of short root in the endodermis where it's not supposed to be expressed does something
very surprising which is it causes many additional layers of tissue to be formed strongly suggesting
that it's supposed to be expressed where it is and there's tight regulation of its movement. So
I would like to thank the members of my laboratory who did all of this work both past and present,
our funding sources the Howard Hughes Medical Institute, The National Institutes of Health,
and the National Science Foundation. If you'd like to learn more about the work that goes
on in our lab you can go to the iBiology series where there are two episodes featuring our work.