9UNP0228SD_Michael_Havey_WordPress

- Welcome everybody to Wednesday Nite at the Lab. I'm Tom Zinnen. I work here at the University Wisconsin Madison Biotechnology Center, and also for UW-Extension, Cooperative Extension. And on behalf of the Wisconsin Alumni Association, thanks for coming to Wednesday Nite at the Lab. We do this every Wednesday night, 50 times a year. Tonight I'm delighted to be able to introduce to you Mike Havey. He's a professor in the Department of Horticulture. He also works for the United States Department of Agriculture in Agriculture Research Service. He's been a breeder of cucumbers since the early 1980s, I believe. Mike and I went to graduate school together. 

He got done just as I was starting back in 1982. He recently completed a sabbatical in Vienna, yes. So I hope you ask him some questions about what it's, is it supposed to be nasty to have to live in Vienna.

- Real bad, real bad.

- You can also see he has a close affiliation with the University in Warsaw. So you have a chance to talk about science both here in Wisconsin and in interesting places like Vienna and Warsaw tonight. When Mike told me what he was going to talk about I was pretty impressed, because I've never heard of any organism that doesn't have maternally inherited organelles. And in this case, the mitochondria are paternally inherited, and that's kind of weird. So I'm looking forward to hearing about his research on how cucumber mitochondria break all the rules. Please join me in welcoming Mike Havey to the lectern here at Wednesday Nite at the Lab. 
(APPLAUSE)

- Thanks, Tom, and thanks everybody for coming out tonight to hear about a plant you probably don't think about very often and that's the cucumber. My talk tonight will be about how cucumber mitochondria break all the rules. Tom mentioned that I'm a USDA scientist. I am that. I work for the United States Department and Agriculture, the Agricultural Research Service, so I'm federally salaried. And we work here at the University. And there are numerous federally salaried, excuse me, faculty here at the University from USDA, the Agricultural Research Service, which I work for, Forest Service. There's also for the USGS, the U. S. 

Geological Survey, among others. Another one is Soil Conservation Service. The unique thing about federal employees here at the University of Wisconsin is that at every other university that I know of, federally salaried researchers or professors have adjunct appointments. UW is unique, as far as I know, in the United States in that we have full faculty appointments here. What does that mean? What that means is we can serve on committees of graduate students; we can serve as major professors. It also means that we had to get tenure just like any state-supported faculty member. That's advantageous for us because if we take a position someplace else we gone through the rigor of tenure. So it's quite a unique situation which I think is mutually beneficial for the USDA. We get to work at this wonderful institution called the University of Wisconsin with all the good biology under way here. 

It is good for the University of Wisconsin because you get essentially endowed or free faculty members contributing to the total community. So it's a very advantageous appointment that I cherish. I also talk about research that I've carried on over the last 10-15 years very closely with Warsaw University, the Life Sciences in Poland. And we continue to collaborate very closely on this research. So when I talk about cucumber, I usually have to introduce the plant because most people don't think much about cucumber. I'm part of a group of scientists here that work on the genetics, breeding and pathology of cucumber. We primarily work on pickling cucumber. That's the cucumber that is in these jars. After the seminar I'm going to invite you up here to try some of the fruits of our labor. A little hands-on experience. 

Pickling cucumber is very unique in that its length and diameter is very well determined because the pickles have to fit in these bottles. And then of course you probably noticed they have the warts or the bumps on them, by botanical term is lenticels. And those are important because when the cucumber is put into the brine, it allows gas exchange and then they ultimately are fermented and they become pickles. If they didn't have the lenticels, they would explode because CO2 would build up inside the cucumber. Pickling is a very old technology developed first in China, spread across Eurasia to northern Europe and why it was so important is it's not only that you can conserve vegetables through the long winters, but it was one of the rare only sources of vitamin C available to people when there were very long winters. The cucumber is not a good source of vitamin C but if you don't have anything else, it is the main source. And that's why it was so widely used throughout northern climates. People when I tell them I work on pickling cucumber the first thing they say to me is that is it possible that you're actually paid to work on pickles? And I always have to point out that whenever you drive down any street in the United States of any community of size, you see a sign like this. And it says billions and billions of hamburgers served. 

Well I like to point out that for every billion hamburgers there's at least two billion pickles that have been consumed, right? Because there's always two pickles on every hamburger. So it is quite a large industry and important and it becomes-- It continues to be important and consumption continues to increase. I will say as we eat more and more fast and convenient foods, pickles are an important part of that total package. There are two other types of cucumbers that we work on. They're down here as well. This one here is called American slicer. You find in the grocery store. It looks something like this. Dark green flesh, they're longer, they're wider. 

Often they're covered with a wax. If you feel that you can feel a little bit of wax cover over them so they don't dehydrate. These are sliced and then eaten in salads. The consumption of the American slicer is going down in favor of this. This is called the European or parthenocarpic slicer. When you buy those in the store they often come in this plastic wrap. This to me is my favorite cucumber. Because it has a very delicate skin, a very soft crunchy taste, soft skin but a crunchy texture and a great cucumber taste. I'm going to invite you up afterwards and if you haven't tried one of these, I would encourage you to come up and try one and then I bet you'll start buying those. These are becoming more and more popular. 

They're called parthenocarpic because they set fruit without pollination. And that's why they're almost invariably produced in greenhouses. And that's why the price is a little bit higher. Whereas the American slicer here has gone through pollination and you'll see some seeds. The result of these parthenocarpic or seedless cucumbers is that the center remains very firm. The texture is firmer and people tend to like crunchy cucumbers. So those are the type of cucumbers we work on. And then our projects work on, as I said, the breeding, the genetics and the pathology to produce cucumbers requiring fewer inputs, such as sprays and fertilizers for production. The second individual that I have to remind you about is the mitochondrion. And if you remember way back in your biology in high school probably you learned about the mitochondrion. 

It's an organelle or a subcellular entity that's carried by all higher organisms above bacteria. You remember it's the site of energy production. It has an endosymbiotic origin and that means is that long ago in the progenitor of the cells in our body, purple non-sulfur bacteria was engulfed by that cell and became a symbiont within that cell. The cell provided substrates and then the mitochondria converted those to energy in the form of ATP. Research on the mitochondria has increased in the last few years because it's been associated, changes in the mitochondrial DNA has been associated with aging, some diseases, especially one macular degeneration of the eye, and then also programmed cell death. In fact, release of cytochromoxidase from the inner membrane here of the mitochondria is one of the hallmarks of program cell death. So there's a lot of research now and interest into the mitochondria. So now I've told you what cucumber and I've told about the mitochondrion. I have to remind you that mitochondria have their own DNA. Now obviously the nucleus has DNA, but the organelles, the mitochondria in our bodies and the chloroplasts in plants have their own DNA. 

That was first discovered in the petite mutants of yeast in France in the '50s where as yeast normally would grow to these big colonies, there were these small colonies that were shown to be due to rearrangements, mutations or deletions in the mitochondrial DNA. The mitochondrial DNA is a circular molecule like bacterium tracing back to its prokaryotic origin and it encodes approximately 20 major proteins, has its own transfer RNAs and its own ribosomal RNAs that translate the RNA to protein. This is an electron micrograph of the circular mitochondrial DNA. Now that original endosymbiont had many, many more genes. But what's happened is many of those genes have moved to the nucleus. And it makes sense. If you have an organelle or a subcellular entity within the cell, and it has its own DNA and you become dependent on its energy production you really don't want that to leave. And so most of the genes from that prokaryotic origin have moved to the nucleus. The ones that are remaining are either too big or have the wrong polarity to import across the membrane so what's left, what genes are left in the mitochondria really have to stay. This is a drawing of your mitochondrial DNA. 

So we're represented throughout the seminar by the Mona Lisa here. Our DNA and that of yeast, a fungus, are very similar. About the same size, about 17 kilobases in size, a circular molecule. And you can see the major genes here are shown in green. Here the ribosomal RNAs. And what happens in your body in yeast is there's one starting point, so from the DNA here an RNA molecule is synthesized all the way around. Every single nucleotide has a coding function in your mitochondrial DNA. The red boxes you see here are transfer RNAs, and they are self-splicing so the RNA will fold upon itself, splice and then each of these individual units will be translated into proteins. So the amazing thing here is that if you look at how diverse from yeast to humans the mitochondrial DNA is very, very similar. And of course, the mitochondrial DNA in the vast majority of fungi, plants and animals is maternally transmitted from the mother. 

That's been well established, in fact, that's the basis of the hypothesis of the biblical Eve in Africa because the mitochondrial DNA in all humans is so similar that it supports the idea that we all trace back to a small group of women in this region in Africa that 100,000 years ago, migration outward to the rest of the world resulted in uniformity of our mitochondrial DNA. Okay, so now I told you about cucumber and mitochondria, the seminar title is, "Cucumber Mitochondria Break All the Rules," so let's go through the rules and see how they break. So for most mitochondria such as here in our bodies versus cucumber, as I said and Tom said when he introduced me, the vast majority of mitochondria from plants, animals and fungi are maternally transmitted. In cucumber I'm going to show you that they are paternally transmitted from the father. That's quite unique. I pointed out that in humans and in fungi such as yeast the DNA is relatively small, only 17 kilobases in size. The cucumber mitochondrial DNA is enormous. It's 1.5 megabases in size, which is just slightly smaller than the bacterium E. coli. So it's really big. 

90 times bigger than humans. I mentioned that in humans there's a highly conserved linear order of genes that's what I mentioned with regard to the out of Africa hypothesis. Cucumber, because there's so much repetitive DNA, so much extra DNA, rearrangements are common and I'm going to show you a unique mutations that are caused by rearrangements in cucumber mitochondrial DNA. And then finally I'm going to finish up with where our research is going now. And that is there is no efficient method to introduce foreign DNA into the mitochondrion. To transform the mitochondrial DNA. And we think that cucumber may be a model for mitochondrial transformation. And we think that's important because if the mitochondrion is playing an important role in programmed cell death, in aging and in some diseases we should be able to correct those by transforming in non mutated or deleted genes. So that's where we're going to go tonight. Okay, paternal transmission. 

Throughout the seminar I'll show you pictures of the people that helped me in this research. This is my first graduate student, Wayne Kennard. In the early '90s, we were working on genetic mapping of cucumber. And what you're seeing here is an audio radiogram that Wayne brought to my office many, many years ago. And what this is is that these spots are just different sizes of DNA. That's all you need to know. The first one here is a size marker. So here's the DNA from this plant and here's the DNA from that plant and you can see there's a difference in that size. Okay, here's the F1 from the cross, a hybrid between the cross between those two plants. And then these are F2 plants from self pollination of an F1 so we would expect if this was in the nucleus that we would see both of those bands segregate in a Mendelian ratio. 

So Wayne brought this into my office and said, look here Mike the F1 and all the progenies have the band of the father. And I said, Wayne you got to be more careful. 
(LAUGHTER)

 You got the two parents mixed up when you loaded them on the gel. First of all a plant breeder always puts the female on the left, and you got that backwards and you just made a mistake loading the gel. Go back and do it. So he did. A week later he comes back to my office with essentially the exact same on audio radiogram. And then I really got mad and I said you got to be more careful Wayne. You made a mistake when you isolated the DNA. 

So go back and isolate DNA, do the experiment over. About a month later he came with the exact same audio rad. Now the morphology between these two plants is so distinctive that we would never make a mistake. So after sending him off and he repeated the experiment three times, remember you replicate everything three times, I started to believe him. So we started following up on this, and a few years later published paper in 1997 showing paternal transmission of the mitochondria in cucumber. And just very briefly, ATP 9, this is subunit 9 of the ATP complex is a well-known mitochondria gene in your body and in the body of all the cells of all eukaryotes. And very simply these are two plants, here's S and C. And you could see when C is the male, you have the C band. When S is the male, you have the S band. And this is a nuclear difference. 

The difference between the two parents, and you can see the hybrid progeny here carry both bands. So that shows that this is paternally transmitted. And we've repeated this with many, many, many different genetic markers. So quite a unique situation. Okay, the second thing that we were interested in was the size of the mitochondrial genomic cucumber. Remember I said it's 90 times bigger than that in your body, it's absolutely enormous. About 1,500 kilobases, just slightly smaller than E. coli. So here's cucumber. And that is not a blemish on that cucumber fruit. 

That's actually a size representation of 90-fold difference between those two pictures. So we're talking about an enormous increase in the size of the DNA. So in the early '80s, Ward at the University of Washington was studying DNA and he was subjecting DNA to density gradient centrifugation. He would just simply sheer the DNA and run it through a density gradient on a centrifugation. If it was heavy, it went to the bottom. If it was light, it tended to stay a little higher up. And what he observed is when he looked at muskmelon, cucumber, zucchini and watermelon, these are all related, they're all horticulture curvets, they're all closely related comparing with P in corn, he noticed that the mitochondrial DNA banded the same for all these different plants but when he looked at the total cellular DNA he saw these bumps here called satellite DNA because it bans out separately. For muskmelon he got a bigger one, cucumber got a smaller, and then zucchini squash and watermelon then got down here and saw none. So he went and studied it further and discovered that when you look at these plants, these are all cucurbits closely related, as P in corn more distantly related. The density was essentially the same. 

The percentage of guanine and cytosine in the DNA was approximately the same. But what you were seeing was huge size differences in the mitochondrial DNA. Melon is the largest known of all plants at 2.3 megabases. Cucumber is 1.5 megabases, and then so on. Most plants are right down here around P about 380. So we study cucumber because it's easier to do crosses in the greenhouse here in the cold north. Melon likes it a lot warmer to that's why we focus on this plant. So this banding demonstrated that there were huge DNA differences, mitochondrial DNA size differences among relatively closely related plants such as watermelon, squash, cucumber and melon. So we asked the question what contributed to that huge increase in DNA size? So we compared among these closely related cucurbits, here again are their size. 

We cloned and sequenced regions of the mitochondrial DNA from these four plants. Oops. This is work of a postdoc in my lab -- who is now on the faculty of the Warsaw University Life Sciences and he is shown here. And what you're seeing here is just a result of the sequencing and a specific region of the mitochondrial DNA. This is a -- which is a model plant and I'm sure you've heard about those in the Wednesday night lab. And then here's watermelon, squash, cucumber and melon. The orange boxes are genes. So you see when you go for a relatively small to an enormous mitochondrial genome, you see fewer and fewer genes. Fewer and fewer orange boxes. You expect that. 

Gene density would go down. There's the same genes in the cucumber genome as in your genome. So it's much, much bigger there's going to be lower gene density. What I want you to notice is that as you go down here you see more and more of these black dots. Those black dots in this program represent repetitive DNAs. These are DNAs that are repeated throughout this sequence. And they vary from 100% to about 50% similarity. So what this reveals are pockets of repetitive DNA spread throughout the mitochondrial. Then he showed those pockets of repetitive DNA are both in forward and reverse orientation. So what that means is you've got repetitive DNAs that are very similar. 

Both that pointed this way and some of them pointed this way. That's important because if they're pointed in the same way they can pair, crossing-over can occur and then you get shifts in the arrangement of genes. And that's what I'm going to show you now. So it works something like this. Let's say this is the circular molecule in the cucumber mitochondrial DNA. Okay, A, B, C, D and E are genes, just genes in the mitochondria. These red boxes are repetitive DNA so they are those pockets of repetitive DNAs that I showed you in the previous slide, these pockets right here. And they're similar throughout the molecule. Okay. So what happens is that those two repetitive DNAs can come together and pair. 

DNA pairs. Okay? So now we have this repetitive region here, pairing with its similar repetitive region here. And the same orientation, that's what we're showing here. If a crossing over occurs, an exchange between the DNA strands, what happens is you end up with two DNA molecules. So we went from one, we had crossing over between the repetitive DNAs, now we have two. We have a subgenomic circle and then we still have that big master circle. Okay? Now if this thing, this small circle doesn't have an origin of replication, it isn't going to repeat, and that's not good. That plant cell is going to die. 

Or the mitochondrion is going to die. If it's got an origin of replication, then it's going to be replicated and it's just going to continue in existence. So now, what would happen, we got the repetitive DNAs here and you can see there's another repetitive DNA right there. So that B, that subgenomic circle carrying gene B can migrate around here by random chance, pair up there, the repetitive DNAs are in the same direction, crossing over occurs. And you come back to the big circle, but now there is something different. You remember in the original circle our gene order was A, B, C, D, E; now it's A, C, D, B, E. We've shifted the genes around. Okay? So that shifting of the genes around is important because these rearrangements can cause problems in the mitochondrial DNA if B moves from a region that's highly expressed down to this region which for some reason has lower expression. Okay? 

And that's exactly what we see in cucumber. So let me show you what it looks like. First let me introduce you to inbred line B of cucumber. And this is what all our research is based on. This is an inbred line meaning it has been self-pollinated, crossed to itself for many generations and that it becomes very genetically uniform. It was developed by Bogustaw Kubicki in Warsaw. What he did is he took an old Polish pickling cucumber, this is Milwaukee Dill Pickles but it looks pretty much like that when it's pickled. And it was from a population called -- and what he did is he self-pollinated that thing for 22 generations. And now it's been carried on to many more generations. All the time selecting for high regeneration from cell culture. 

So he was able to regenerate plants from cell culture. That's why the Poles developed this inbred. So over the years they've taken inbred B, through cell cultures, leaf callus direct regeneration, protoplast cell, primordia. These are all techniques to asexually, to propagate plants through cell culture, very common techniques that are used. But what the Poles observed was they kept getting back what they called the mosaic phenotype. What we call MSC for mosaic. And it appears after cell culture. You take that inbred, no variation in the nucleus, in the nuclear genome, pass it through cell culture and you recover these mutant phenotypes. These are wild type cucumber seedlings. These are MSC. 

And you can see how they're deformed. These plants are sick. Here's a wild type true leaf and here is an MSC leaf. And you can see why they called it mosaic. So this is work done by my colleague in Warsaw. This is a picture of Stefan. What he did is said, okay, I generated mutants from tissue culture and that's nothing new so he studied the genetics of them. Okay, so he crossed B, that's what went into tissue culture, with MSC, that's what came out of the same tissue culture, and when MSC is the male, I have it in red here, you'll notice that all the plants are MSC. When MSC is the female, the plants are wild type. And so he published a paper in 1976 describing this paternally transmitted phenotype, at the exact same time I published a paper showing the mitochondrial genome was paternally transmitted. 

So I was fortunate enough to spend a sabbatical leave working with Stefan in Warsaw and we studied this further. And what we undertook was a whole series of studies to try and determine if the MSC phenotype were due to mutations or rearrangements in the mitochondrial genome. We did a whole series of techniques that I won't go into, southerns, northerns. We cloned and sequenced areas. We did westerns, looking at protein accumulation, a whole series of techniques used in genetic analysis. This is one of my students, Jason Lily, who did his PhD thesis on MSC. And what he observed is when he looked at the mitochondrial DNA of the mosaic mutants, he saw differences. Here's the parental B, here's the MSC that came out of a tissue culture, these are other lines I won't talk about. But you can see there are differences, unique here to MSC. Here, not unique to other tissue cultures lines. 

In some cases there were pieces of the DNA that were missing. So the genome had rearranged and in some cases we were missing pieces. He continued on and showed that the rearranged regions had lower expression. So in some cases the genes were there, it's just that they had lower expression levels. And that's what you're seeing here. This is a northern blot. This is one of the genes that it causes a certain mosaic or one of the mosaic phenotypes. And you can see here relative to B there was less, you see less intensity here, lower intensity. And that's because this gene has been through rearrangement, put into an environment where there's less transcription. Less expression. 

And then down here is just a loading control showing the same amount of RNA in each line. Okay, so now I've told you that cucumber mitochondria breaks these rules at least, it's paternally transmitted, it's big, 90 times bigger than in your body and passage through cell culture reveals these unique rearrangements that are associated with mutations in the mitochondrial genome. So right now, I hope you're asking, so what? What difference does that mean? Does anybody know who this man is? A very famous faculty member here, retired in 1963. Anybody? This is John Charles Walker, who was a very famous pathologist here. And when I was a graduate student I learned this quote. "Our research has to be good but it has to be good for something." 

Now I'd like to tell you what I think, what we can use these mitochondrial mutants and the unique mitochondrial genetics of cucumber for something useful. The first thing is that I think what we've proposed and are following up on is that the MSC mutations are a unique way to alter the expression of mitochondrial genes. Geneticists always use mutations. Mutations allow us to learn about specific genetic and physiological processes. There is no efficient way to mutate the mitochondrial DNA. And we think that this is one way to generate mitochondrial mutants. We believe that because not all MSC lines are identical. It could happen that you would have just a few strange molecules among the whole population of mitochondria and you would sort those out. But with the Poles we've generated many MSC lines and they are phenotypically different. I think you'll agree that MSC16 is the basis of all the research I've been telling about so far. 

But I think you'll agree that MSC281 looks different. And that whatever is sorting out of here is lethal. And this phenotype is paternally transmitted so we know it's associated with the mitochondrial genome. So with the Poles, we've taken the wild type, normal and inbred line B, remember that's highly inbred, passed it through different types of cell culture. Here's direct regeneration of plants from leaf callus. Shoot primordia culture. These are regenerated from protoplasts. And these are regenerated from salt-resistant callus. Just tissue culture that's been grown in high salt concentrations. And the phenotypes, the appearance of these plants are quite different. 

For example, here you see one type of fruit different from the original. Here you see a different color of the fruit, lighter. Here you see again the sorting. These traits are all paternally transmitted. So they're associated with the mitochondrial genome. If you look the DNA of these plants, they're quite different. And this is a very busy slide. But what I'd like you to notice is that when you look at across the top, here is B, this is the wild type highly inbred line that went into culture and if you look at the lines here these are just different types of culture. These are regenerates from those different types of cultures. Here are four regions of the mitochondria genome and you look across and you just notice those differences. 

It's not all the same rearrangement. Sometimes they're unique, sometimes they pick up out of the cell culture, you get the same thing again. Sometimes they're different from all the others. So the mitochondrial rearrangement are not all tracing back to just a few that exist. We're getting different ones out of different cell cultures at different rates. So we feel that that's a way that we can mutate the mitochondrial genome. Now they're not really mutations. I'm sure you've heard it Wednesday Night Lab about knock-outs. When you knock-out a gene, mutate it to non-function. If he mutate mitochondrial genes to non-function, you're dead. 

So what we do is we knock them down, meaning that there is expression but it's at a lower level allowing the plant to survive and then we can study what effects, for example, what changes occur in the nuclear genome in response to these it mitochondrial knockdowns. So we're working with the Poles to do exactly that for a set of independent rearrangements and phenotypes that we've generated over the years. And finally I'm going to finish up with where our research is going now and that we think cucumber offers a unique opportunity to introduce foreign genes into the mitochondrial DNA. And we're hoping that we can develop it as a model plant for mitochondrial DNA transformation. Why? Cucumber has one other unique attribute I haven't told you about. And that is that in microspores which are just immature pollen, the male gamete of plants, cucumber microspores possess a few huge mitochondria. So what you're looking at here is an electron micrograph through an immature pollen of cucumber. Here's the nucleus, I hope you can see the nuclear envelope there. See these large circles here? 

I hope you can see it with the lights this bright. Those are mitochondria. Normally the mitochondria would be about the size of that white dot right there. But these things are huge. So then they become an obvious target for ballistic transformation to shoot in foreign DNA into those large mitochondria. This is the gene gun, simply put all you do is these are gold particles, we coat DNA onto the gold particles, put them into the gun, put our microspores, our immature pollen underneath, fire them in, they shoot in. What do they shoot into? We're aiming for those mitochondria right there that are big. And then will be transferred to the progeny because remember they're paternally transmitted. Can we shoot into microspores? 

We sure can. When I was on sabbatical in Vienna, that's one of the experiments I completed. And what you're seeing here in these are microspores, immature pollen of cucumber, we just use a reporter Gene, called Gus. The condition is this blue color. So we fired them in. This is just expression but you can see that it's expressed so that we know we can get them in and that it's expressed. I'm not sure if you can see it. I can't see from here. But these are the gold particles if you can see right there and right there, that's the size of the gold particles. So you can see they're much smaller than the microspore but they go right on in. 

So what we're working on now, and I just got funded on a grant to have a postdoc work on this, is the idea is we'll take the microspores with the huge mitochondria, ballistically transform, shoot DNA in to the mitochondrion and then either take those microspores and mature them to pollen and use them in crosses to a female plant. Mitochondria are paternally transmitted so those transformed mitochondria will go through what we call the gametophytic stage. The gametophytic pathway to the progeny. Or we could induce embryo genesis, produce embryos which is the sporophytic pathway and regenerate plants directly. And either of those we should be able to recover a mitochondrial transforming. And that's the work that we're doing now. While I was on sabbatical last spring in Vienna, I worked with a colleague who works on both of the gametophytic and sporophytic pathways in plants from microspores, and we were able to generate pollen from microspores, mature them in vitro. And here's one that's gone towards the sporophytic, because you can see there are two very clear nuclei there. So it is possible to do this but we have to work on now is get the numbers high enough so we can regenerate viable pollen or plants. I can tell you now that I don't think this is going to work. 

Because the number of pollen that mature from the microspores is low enough that don't think the probability is high that we're going to capture the transformed mitochondria and transfer them into the progeny. And so our research is now focusing on generating embryos from the microspore and then regenerating plants. Okay, I'm going to finish up. It's a quarter to. So cucumber, we think it's a model for organellar genetics because paternal transmission allows separation of chloroplasts and mitochondria. Chloroplasts, the other major organelle in plants, is maternally transmitted in cucumber. The mitochondria are paternally transmitted. So we can separate those two. Recombination among repetitive DNAs in the mitochondrial DNA of cucumber causes these rearrangements that affect mitochondria gene expression. We think that's a way to mutate to the mitochondria genome through these knockdowns. 

And that the large mitochondria in microspores may allow for mitochondrial transformation. I'm going to finish up by giving special thanks to a whole group of people. As I said, Wayne Kennard and Jason Lily were graduate students in the program here. I do have to thank my employer USDA and then the wonderful University of Wisconsin that allows me to work here. I work very closely still with Warsaw University, the Life Sciences, Stefan, and others. We've enjoyed funding from a whole series of groups from primarily NATO and the USDA. And to finish, do remember the old John Lennon song "Give Peace a Chance?" So what I'm asking is when we're done come on down here, try some of these cucumbers and please give cucumber a chance. 
(LAUGHTER)

 Thank you. 
(APPLAUSE)

- Would you like to answer a few questions?

- Sure, sure. Any questions from anybody? Yes, sir?

- What happens to the female mitochondria?

- Yeah, the question is a very good one that we've been struggling with for a long time is what happens to the female mitochondria? To answer that, but let me tell you first of all, we don't know. But let's back up and look at animals and fungi and plants under normal circumstances. If you think about the egg cell is enormous compared to the sperm cell. 

Okay, the sperm cells obviously have mitochondria because there has to be energy to swim or to go through the fertilization process. So the idea is that you have so many mitochondria in the egg cell, that after the two gametes unite, that maybe there's just the male are so few that they're just washed out, they drift away. If that's the case how is it possible that an egg cell, a cucumber which is enormous compared to the sperm cell has paternal transmission. So there must be an active process to either select for the male mitochondria or degrade the female mitochondria. And one of the reasons why we want to develop mitochondrial transformation is to answer that exact question because what would like to do is transform the male mitochondria with one fluorescent protein and the female with another fluorescent protein. Allow fertilization to occur and then ask the question what happens to those mitochondria? Are they all pushed off to the side? Do they disappear? It's very difficult to do that experiment with only transforming the nuclear genome because then you would have it expressed in the hybrid. And so we haven't been able to figure out a way other than that to answer that question. 

But there has to be, in my mind, some active process either discriminating against, degrading those female mitochondria. Yes, ma'am?

- Does any of this work have anything to do with an agricultural application in relation to cucumbers and how they grow and change?

- The question was how does this research relate to agricultural production? Actually that's one I didn't go into that at all. But many hybrid plants and hybrid crops are generated using systems that are known as cytoplasmic male sterility. And that is, the conditions male sterility in the female line so that all pollination comes, so it's out-crossed. CMS or cytoplasmic male sterility is conditioned by the mitochondria genome. One of the reasons we've undertaken this research is that if we can introduce foreign genes into the mitochondrion and we can very efficiently develop CMS systems which would have great applicability in hybrid seed production, so that's one of our long-term goals. Actually it wouldn't work in cucumber so it's more model because cucumber is what's known as a diclinous or monoecious. 

It has male and female flowers that are separate. So these hybrid cucumbers and these are all hybrid, they're controlled so that you have a female plant with only female flowers and a male plant with only male flowers. And that produces the hybrid so the CMS system wouldn't work in cucumber but it would work in other plants. So yes, there is direct agricultural use of these technologies. Yes, sir?

- The large mitochondria and microspores, they seem to be very disproportionate. Is this common in other plants and what's the evolutionary cause, do you think, of such large mitochondrial DNA in those particular families of plants?

- That's a wonderful question. The question is why do we have big mitochondria and why do we have a big mitochondrial genome? There has to be some reason evolutionary advantage to that that would cause that to happen. 

And then you also asked is it common in other plants that you find these big mitochondria in microspores? So I've looked through the literature and looked through many pictures of microspores in other plants, I've never found any other example of large mitochondria in the microspore of any other plant. Now it could exist because people might not have looked. But there's no evidence. But is a real quandary that I've thought about for a long time. It just doesn't make sense. Why do you have paternal transmission, large mitochondria and then relatively few in the pollen. And I'll give you my explanation which I have no data for, so is all hypothesis. What I think happened, and I'm not sure it's kind of a chicken or the egg thing, what came first, but if you think about it let's say that repetitive DNAs got into the mitochondria DNA and they started increasing. You have lots of repetitive DNAs in your body. 

So over time genomes can get bigger and bigger and bigger. So if the mitochondrial genome got bigger and bigger and bigger and you started having a recombination causing changes and shifts in the genes that I showed you, and the mitochondria is strictly maternally transmitted you're going to accumulate knockdowns, knockouts. They're going to be selected against but remember in every cell you have many mitochondria and in every mitochondria you have many copies of the DNA. So most of them are just going to disappear. But there could be problems and that's actually one of the hypotheses with regards to some aging and mitochondrial inherited diseases is due to these rearrangements. So you get this mitochondrial genome that it's bigger and bigger and bigger, starts rearranging and all of a sudden, you're finding the genes aren't working, you're losing them, and they're not functioning quite as well. And if it's strictly maternally transmitted it might be hard to correct that. You could evolve a nuclear system to correct it and that is known. But wouldn't it be efficient to send it through the pollen, send it through the male? When there is relatively few mitochondria? 

So take that microspore, it's got these big mitochondria. You can count there, I don't know maybe I should have counted up, maybe there's 12 or 15. Normally there would be hundreds of them. Now how many of those are going to be grabbed by the sperm cell and transferred to the progeny? Very few. Very, very, very few. And that's the bottleneck that we see that occurs when we sort these mitochondrial mutants. So my hypothesis is that the genome got bigger and bigger and bigger and then this is a method to put it through a bottleneck to select those that work. Now remember, like in humans, plant cells pollen lands on the female flower, a pollen tube has to grow all the way down. The sperm cells have to be delivered, -- or fertilization has to occur and then it develops. 

So I'm wondering if this is a bottleneck to sort out, to get rid of those non functioning mitochondrial genomes. That's the best explanation that I've been able to come up with and if anybody's got another one, I'd love to hear from you. So think about it over the weekend and call me on Monday. 
(LAUGHTER)

- What's known about the function compared to other mitochondrial -- ?

- RNA or DNA?

- Whatever it is that's copying the DNA of the mitochondria.

- The question was what about polymerases in the mitochondria in general, and in cucumber in specific.

- Do they make more mistakes to make up different structures? Is there some way that they're different from other mitochondria?

- Do they have a different structure or function? To my knowledge, all the polymerases, the DNA and RNA polymerases that function in the mitochondria are nuclear encoded, cytoplasmically translated and imported into the mitochondria. And they're classes of special DNA and RNA polymerases that are common among plants, animals and fungi. To my knowledge they're different from the nuclear DNA and RNA polymerases but they're very similar among some very distantly related eukaryotes. I have no evidence in cucumber of them being any different.

- I thought that the mitochondria had its own polymerase.

- They do have their own polymerases but they're nuclear encoded. So they're different from the nuclear, they're nuclear encoded, cytoplasmically translated, and then specifically imported to the mitochondria. So they're not encoded by the mitochondria genome, per se. To my knowledge, we haven't looked at all of them, but to my knowledge I know of no major difference in those polymerases. 

So to me the mitochondria in cucumber is working just like any other plant and it just has these different characteristics.

- Another question.

- Yes, sir?

- Are there special challenges to sequencing the genomes of mitochondria?

- Are there challenges to sequencing the mitochondrial genome? Not really. It's easy to spin down the mitochondria, purify the DNA, clone it and sequence it. Cucumber tends to be very AT rich that resulted in that special satellite band in the density centrifugation experiments. But nothing unique. So just nobody's done that due to the cost and as the cost comes down, some day it will. 

But it's just big enough that we only sequenced pieces of it.

- Can you isolate the mitochondria and use them in a system to see whether the size of the DNA affects the efficiency in terms of converting and in terms of energy production?

- The question was is there any differences in amounts of energy production, methods of energy production of the mitochondrion of cucumber.

- -- compared to some other mitochondria that the don't have these large structures is there a difference in the amount of ATP produced --?

- I don't think, I don't know and anybody who's ever studied that. But mitochondrial genome of cucumber has no additional genes that we know of. We've not been able to identify any unique coding regions. And in normal plant tissue the size of the mitochondria are the same as other plants. So it's only these big ones are only found in the microspore. When you go to the plant, they are normal. 

But for example measuring actual ATP production, I don't know of any research on that per unit, per number of mitochondria or something like that. So I wouldn't say there's any evidence for them being any less efficient except in the MSC mutants. That has been studied and they do have a deleterious effect on mitochondrial function across the board. Which makes sense because you have much less expression of a very imperative gene because, as I said, there are 17 genes in your body, there's about 23 in the cucumber so there's very few genes that are actually remaining in the mitochondria. And as I said, those are imperative for mitochondrial function.

- Is the repetitive DNA is DNA that carries the same codes that are in other parts of the genome that are repetition of genes elsewhere on the mitochondria genome.

- The question was about repetitive DNAs in the mitochondrial genome.

- And is it possible that the advantage of having repeats is that at some point the organism was subjected to some kind of radiation or something like that so you have one target that didn't get hit with --.

- So the question is the GC content, the repetitive DNAs tend to be very AT rich. So you have long stretches of AT, AT various mixes of As and Ts. 

The coding regions are GC rich. So in the original density centrifugation they showed that the mitochondrial genome among a large number of plants is about 45% GC. So the AT rich regions, the coding regions are 45% GC. The AT regions are obviously lower. The codon usage, so what codons are used for specific amino acids in the mitochondrion are not different from any other mitochondrion. So there's no evidence that the DNA is somehow different other than the amount of repetitive DNAs. So these pockets of repetitive DNAs.

- Any other questions?

- There was one in the back?

- Well, if you think of one, come on down. 
Let's thank Mike. 
(APPLAUSE)