My name is Philip Benfey I’m a professor at Duke University and an investigator with Howard Hughes Medical Institute Today I’m gonna give you an introduction to root genetics This is Part 1 of a two-part series In the second part, I’m gonna go more in-depth into the research that my laboratory performs, and most of it is on root development and root genetics So, I think it’s first reasonable to ask, why study plants at all? Why not study something that will be more directly related to human health? And I would present to you the fact that plants are really at the basis of some of the most important challenges facing the planet today An example — perhaps the best and first example — is climate change Climate change is having a major effect on agriculture, that is… either when there’s too little water, in droughts, too much water, with floods But also, I would… I would point out that plants can play a major role in mitigating climate change Plants are where that the leaves take up carbon dioxide to perform photosynthesis That carbon is then put into roots, a place where they could you could sequester carbon, take it out of the air, and put it back in the ground where it came from Plants are also at the… can are part of another way to address a major challenge for the planet, which is hunger, human hunger There are still many cases of malnutrition, and plants are the major source of food for the planet Third, transportation fuels — energy security Plants are a potential source for providing liquid fuels, the sort of fuels that are really the only real option, at least in the short term, for things like jet travel Now, when you’re studying plants, you can obviously study the aerial parts of the plant, that is, the above ground, the green parts And I’m gonna tell you about studying what it means to study roots In fact, for most people, in general, roots are really thought of as… very little They’re out of sight, so people don’t think much about them, so it’s out of sight, out of mind And yet, roots play really, really critical functions for plants The roots are the major location they’re the major place that plants acquire water, a critical feature of plant growth They’re also the place where they they’re where plants pick up nutrients, that is, nitrogen, phosphate, potassium — all those things that plants require to grow And perhaps something you don’t think about too much, but roots are what keep the plants upright They are what anchor the plant And so, if the roots are shallow or not well placed in the earth, with the first wind, a plant will get blown over And last but not least, roots are where there is the major interaction with microbes, many of these microbes being bacteria, in particular, and many of these can be beneficial The bacteria are not necessarily pathogens They can be beneficial, as shown here, where these are nodules that form on soybean roots, and those nodules allow the plant to take up nitrogen and add nitrogen to the soil So, plants are unlike humans or animals, where the embryo has almost all of the features of the adult In a human embryo, you can see already the head, the arms, the legs, etc Plants, on the other hand, have are much more potential when they come out of a seed or are in the seed And that potential is at the tip of the of the embryo that and the base of the embryo So, when the seedling starts to grow, it has a source of stem cells, cells that have the potential to make all the other cells of the body And there are cells at the tip, which is called the shoot apical meristem There are cells at the root tip So, the shoot tip and the root tip And the root apical meristem is what makes all of the cells of the root We decided to work on roots primarily because, from a developmental perspective, from asking questions such as how you go from a stem cell to a fully differentiated tissue, roots have some great advantages over even other parts of the plant, and certainly over animals If you look at the root, it actually… if you if you look at the right side, here, they are the cells are organized essentially as concentric cylinders So, there’s this outer cylinder, a next cylinder in, etc

And if you move that root around, you have radial symmetry So, that’s really straightforward So, taking a line from the center of the root outward, you get all of the different cell types of the root That root… that line can be drawn in any direction, and you’re still getting all of the same cell types Now, at the very tip of the root, as I mentioned, there is set of stem cells What are stem cells? Stem cells are cells that have the potential to regenerate, and also to give more than one cell type — different cell types — after they divide And these… there are four different stem cell populations at the tip of the root, and they give rise to every other cell population in the… in the root itself So, to understand how we would go from stem cells to fully differentiated tissue, how we… how the root or any organ gets organized so that the cells that are in the right places and have the right identities, we’ve taken a genetic approach Genetics was essentially invented, or discovered, by Gregor Mendel, who was a monk working in what at that time was Austria And in his garden, he would look at peas, and he noticed that there were peas that had different colored flowers, different length of their seed pods, etc To translate that approach to roots, we had to work out a way to grow roots in such a way that we could see them — we could see their differences And so, for that, we’ve been working on the plant that is a member of the mustard family It’s called Arabidopsis thaliana It’s a small plant And we grow it on square petri dishes These petri dishes are filled with agar that is somewhat similar to Jell-O And in that agar, there are also the nutrients they need: nitrogen, phosphate, etc Now, on the left, you see a wild type plant, that is, the normal laboratory plant, and you see the length of the root at this time And then, on the right, you see plants that were, we discovered we identified as being mutants And originally, we thought that they were the major and most important feature of these plants was the fact that they had shorter roots So, we named them short-root, and that’s what you see in the middle here And it was only later, when we took serial sections — we took transverse sections through the root — that we noticed that, in fact, these roots had a very striking anomaly There was something very strikingly different about them, as compared to wild type So, if you see on the wild type, on the left, you see in the outer level of epidermis the outer layer of epidermis Inside that, there are eight cells that form the cortex Inside those eight cells, there’s another layer of cells called the endodermis — again, eight cells looking at that transverse section at the top And inside that, there are smaller cells that constitute the vascular tissue Now, in short-root, when we looked at the section between the outer layer of epidermis and the internal small cells of the vascular tissue, there was only a single layer So, there’s a layer of cells that is missing here We later found another mutant, that we called scarecrow after the character in The Wizard of Oz, who is missing a brain We realized it was missing a cell layer, so we were looking for something that was missing an aspect, like missing a brain for scarecrow And here, again, we find that there’s a single layer between the outer layer of epidermis and the internal layer of the vascular internal layers of the vascular tissue Now, how do you make those two layers, the endodermis and cortex? Well, that green cell is a stem cell And it divides first along the transverse axis to regenerate itself As I mentioned earlier, all stem cells have to have a regenerative process so that they can maintain their stem cell properties The cell above it, then, the daughter of the stem cell, divides along the longitudinal axis to give the first two cells of those two lineages, the endodermis and the cortex — the blue being the endodermis, the yellow being the cortex And so, when we looked at our mutants, short-root and scarecrow, we said, well, they only have a single layer there, so that second division must have gone wrong And so, at this point, I let me explain the key leap of faith of genetics In genetics, we have a belief — and it’s been shown to be more than a belief, but really to be a fact — that if you can figure out what’s gone wrong in the mutant, that tells you what the normal — what we call the wild type — gene is doing in its normal context

So, here we realized that if that second division is missing, then both genes, both short-root and scarecrow, must be required to get that second division to occur And then the question arose, well, what is it that is… that remains? If that second division hasn’t occurred, does that mean that it’s produced endodermis? Has it produced cortex? Or has it produced some combination of the two? And so, using a number of different antibodies, etc, to look at attributes of either cortex or endodermis, we saw that in short-root what had happened was that we had all the attributes of cortex but none of the attributes of endodermis Well, in scarecrow, we actually had attributes of both cortex and endodermis And so, to interpret this, what we said was, well again, we’re using that great leap of faith of genetics, saying, well, if we know what went wrong, what is it… what ha… what does the wild type gene product what does the gene in its natural state actually do? And again, there are three things that have to happen here There has to be the division There has to be specification of endodermis, and specification of cortex And so, in short-root, there are two things that have gone wrong The division has not occurred, and endodermis has not been made, so that tells us that the short-root gene product has to do two things It has to make the division and specify endodermis Well, in scarecrow, because we see both endodermal and cortex attributes in that cell, it suggested that the primary function of the short of the scarecrow protein in this context is just to make the division After identifying the probable function of the genes, we wanted to identify the actual genes themselves And so, using a number of different approaches, we actually… what we say… call cloned the gene, that is, identified the actual gene that had been mutated And we use the promoter of that gene the promoter is the part of the DNA that regulates expression, that causes the gene to be expressed in a specific place in the plant And we asked, is this gene expressed where we thought it would be? That is, is it expressed in that cell that has to divide along the longitudinal axis and perhaps continues to be expressed in one of the two layers? And in fact, what we found was that it’s expressed exactly right here That is the tis… the cell that has to expre that has to divide to give the endodermis on the inside, the cortex on the outside It turns out that scarecrow remains expressed in the endodermis, and that for reasons that we later discovered We then looked to see about see if the scarecrow protein was expressed in a similar manner Now, to do that, we used the same promoter — that same part of the DNA that regulates expression We now have it driving the coding sequence, that is, the region of the DNA that codes for the scarecrow protein And we fused the scarecrow protein, now, to GFP itself So, now we’re looking at the actual protein e xpressed in cells And again, what we see is that it’s expressed exactly where we had expected, here This is the stem cell that has to divide It remains on in the endodermis, all the way up in that lineage And so, for scarecrow, it was a very well behaved gene It expressed in exactly the cells that we expected and a few more, but that didn’t make any difference And then we st… then we looked at short-root Now, in short-root, we had a surprise When we looked at the short-root RNA so, again, on the right-hand side we see short-root promoter driving GFP So, this is where the RNA would be expressed But just to be sure, we also did something called an in situ hybridization, where we actually used the short-root RNA itself and probed the actual a real section of the root to see where the RNA is And in both cases, we said we found that the RNA was not where we expected The RNA in this case is in those small cells in the middle of the root This is the vascular tissue It’s not where you would expect it, which would be in these cells over here So, what’s going on here? Well, it… it wouldn’t be too hard to explain So, an explanation that if this were an animal, the most likely explanation would be that short-root perhaps is a transcription factor — that’s what we later determined it to be A transcription factor is a protein that binds to DNA and causes it to be expressed causes genes to be expressed in some location So, one could imagine that short-root goes into the nucleus, turns on another gene,

that gene makes a small peptide, for example, that moves between the two cells, and there’s a receptor on the second cell, that then sends a signal down, and that turns on other genes, here In plants, there’s another pathway, though It is possible to actually move physically from one cell to the next, to have proteins move between one cell and the next, through channels that are called plasmodesmata And the evidence is that that’s exactly what’s happening with short-root, because when we used the short-root promoter driving the coding sequence of short-root, which was fused to GFP — so, we’re now looking at where the protein is — what we see is the protein actually right where we expect it It’s in the stem cell And the protein remains in the cells Each one of these endodermal cells has the protein there Notice that the protein actually is found in the nucleus That’s that little ball of green inside the red outer edge of the cell So, that is consistent with short-root being a transcription factor Transcription factors, again, bind to DNA They do that in the nucleus of cells In fact, short-root and scarecrow are members of the same family of transcription factors Now, so… why then do this strange process, where the protein physically moves from the vascular tissue into the next layer over? Why not just express this factor, this short-root protein, in the endodermis itself? And so, we decided to ask, what if we changed the expression, that is, caused short-root to be expressed in the endodermis? What would happen? And to do that, in this case we used the promoter of the scarecrow gene fused to the short-root gene So, we’ve now used we’re going to express short-root, not where it normally is in the vascular tissue, but in the endodermis, where scarecrow is expressed express it alongside scarecrow And what happened, then, what we found was, compared to the upper sid the upper panel, here, which is the normal root, which has the two layers endodermis on the inside, the cortex on the out in this case, we see lots of additional layers So, by putting the protein into the endodermis, we cause a massive expansion of the number of layers in the endodermis And thus, this may be part of the reason why we have this complex process of short-root being expressed in the vascular tissue, and only moving one layer over In my second talk, I will go into more depth about how this is done and why short-root doesn’t move further in the normal situation But now I want to talk about another aspect of roots, another reason why we decided to work on roots over 25 years ago So, I’ve already mentioned that there is this radial symmetry, which simplifies asking questions about how cells get specified, how you pattern the root Well, another simplifying feature of the root is that the stem cells, as I’ve already mentioned, are at the tip of the root Now, as you go up any one of these what we call cell files, that actually corresponds to the age of the actual cell, or its developmental stage So, as we’ve seen, the stem cells divide The cells that they produce, then, are the first cells of those lineages And those cells will then go through divisions, etc Along that cell file, the youngest cells are at the tip of the root, the older cells are at the upper part of the root So, what does this mean? It means that at any… at any one time, if we look along a root, we have all of the developmental stages, from stem cell to early cell, more developed cell, more differentiated cell, all the way to fully differentiated cells Now, in the endodermis — something we’ve been discussing, that cell layer we’ve been discussing, that is missing from the short-root mutant, for example — there is a signature differentiated feature It’s called the Casparian strip The Casparian strip acts as a barrier It acts as a barrier to water So, water actually when it comes from the outside of the root, can go between the cells, all the way into the vascular tissue unless there’s a Casparian strip So, the Casparian strip is actually a waterproofing that goes around the endodermis, each of the endodermal cells And it’s blocking water from coming in It gets stopped there

And so, that means that the outer layer of the endodermis has channels to allow only a certain amount of water through, and also whatever it is dissolved in the water — nitrogen, phosphate, etc That has to be… can be selectively taken up before it gets into the vascular tissue You can see these this Casparian strip on a transverse section They look like… when stained, it looks like these little red or orange points around the root And what this is… what the staining is is something called lignin This is a waterproofing that is made by the plant, only in these very specific locations Where the Casparian strip is localized is dependent on something called the CASP proteins This stands for Casparian strip associated protein These were identified by Niko Geldner as proteins that localize the Casparian strip along the membrane of the endodermis These can be marked with GFP — that is, GFP, green fluorescent protein — that’s fused to the coding sequence of the CASP genes, and this is a good marker for differentiation, then We know that these CASP proteins are turned on only in one location in the endodermis, and only when the endodermis is becoming terminally loca terminally loc… differentiated And so, we decided to perform a genetic screen And this time we’re asking for genes that, when mutated, affect the expression of the CASP proteins And so, these could be — hopefully — regulators of the end stages of differentiation Previously, with short-root and scarecrow, we identified mutations that affected the very early stages of stem cell division Now we’re looking for genes that affect this very end stage of this process of going from stem cells to fully differentiated tissue And what we found was we did this screen by looking under the microscope So, individual plants were placed under the microscope, where, on the left you see this is what the normal plant looks like This is the CASP protein localized, in this case, to the nucleus And then, we were looking for things that look like this That where… either the CASP protein was turned off entirely, or possibly was… changed its localization And what we found were mutants that actually had completely wiped out the expression of these CASP genes And when we characterized them further, they turned out to be another transcription factor called MYB36 And we asked, well, now that we have something that changes the last stages of differentiation, can we use it or maybe short-root, which stained we know is involved in the whole differentiation program of endodermis can we use these to change a tissue that has a totally different program In this case, we’re talking about the outer layer, the epidermis And we asked, if we express short-root, by using a promoter that functions only in the epidermis, or this MYB36 in the epidermis, can we cause it to become endodermis? Is that sufficient? Is it enough to just take one transcription factor, and cause it to be expressed in a different tissue, to completely change the identity of that other tissue? When we tried MYB36, it sort of worked Not… it was not very effective However, when we tried short-root, we did find that when we put it in to the epidermis — when we caused short-root to be expressed in the epidermis — in this case, we found evidence for for the CASP genes to be turned on, now, in the epidermis, as well as lignin So, actually, evidence for a Casparian strip There was a problem, though, as shown in this movie, that what you’re looking at is the CASP expression turning on And you notice that it’s not turning on in every cell It doesn’t even stay on It turns on and turns off And so, we couldn’t quite understand why we couldn’t get it on in every cell We knew short-root was being expressed in every cell of the epidermis, but why was why did we get terminal differentiation only in a few cells? And it wasn’t even terminal; it lasted for a while and then turned off again And so, what we found was that there was something missing And what was missing was a small peptide A peptide means about 20 amino acids, so it’s not as big as most proteins, just a very, very small number of amino acids

that form something that interacts with a receptor to cause something to happen What happens, as shown again from Niko Geldner’s lab was that this protein is made in the vascular tissue; it goes from the vascular tissue over to the endodermis; and there, it causes the Casparian strip to fully close The Casparian Strip in the absence of this peptide has little holes in it When this peptide comes across, it causes it to completely close And we wondered, well, what if we add this peptide to our plants that are expressing short-root in the epidermis? Will we see any change? And in fact, we saw a dramatic change So, now when we added this peptide, CIF2, we see almost every cell now has a Casparian strip The surprise was it’s and not every cell in the outer layer, but every cell in the next layer under this Now, there are multiple layers here, again A little like when short-root is expressed in the endodermis, when it’s expressed in the epidermis, you also get multiple layers And it’s only the next layer in, the subepidermal layer, that now has Casparian strip all the way around it And so, we have really been able to change the identity — completely change the identity of a cell layer — to a… what looks like a fully differentiated endodermal cell by expressing one transcription factor, short-root, and the addition of this peptide hormone, CIF2 So, let me summarize what I’ve gone through in this introduction to root genetics First, we talked about studying plants actually could help define solutions to some of the absolutely most critical issues facing the planet today I also pointed out that roots play really important roles in both plant growth and health, but up until recently, it’s been difficult to study them Now, by using new approaches, by looking at plants grown outside of soil, we can do genetic screens for mutants In our case, the first one we did was a very simple screen, just looking for mutants with shorter roots And we identified two: one that we called short-root, and the other one scarecrow And it turns out that both of them are actually missing a critical cell layer What I then showed you was that short-root protein physically moves from one cell layer into the next, and that movement is critical for its function Then we showed that if you put short-root in where it’s not supposed to go and the first time we put it in the endodermis — caused it to be expressed in the endodermis — and that dramatically changed the pattern of the cell layers in the root, making lots of ectopic cell layers Then we did a genetic screen for changes in the terminal differentiation pattern and identified MYB36, which has a really important role in controlling those end-stage differentiation functions And finally, I showed that expression of short-root in the outer layer, in the epidermis, combined with a peptide factor, can lead to a complete change in cell identity This is an image of my laboratory, the people who did the work that I talked about eating at a nice local restaurant Those are our sources of funding for this work And I want to point out that this is Part 1 of two parts, where in the second part I go into much more depth as to how these different factors, as well as other genes that are involved in root development how they function and how roots actually form and explore their soil environment