To understand how superweeds survive, we first have to understand why normal weeds (the Jimmy Olsens and Lois Lanes of the plant world) die. <– last superhero reference of this post I promise.

Plants are not like people. The list of differences goes on and on, but today the difference we’re concerned about is where amino acids come from. Amino acids are the building blocks of proteins, the same way Adenine (A), Thymine (T), Guanine (G) and Cytosine (C) are the building blocks of DNA. Both our bodies and plants (and almost every other living thing) use the same twenty amino acids to build proteins. Our bodies can make ~12 of the twenty animo acids for themselves, but there are at least eight amino acids that the human body cannot produce (called essential amino acids). Our only source of these amino acids is from protein in our food.

It’s all well and good for us to get amino acids from our food, but plants don’t eat. They’re made of pretty much nothing more than water, sunlight and air. And trust me, none of those things are a good source of protein.

Unlike us, plants have to be able to make all twenty amino acids from scratch. That means they need whole biochemical pathways* that aren’t found in animals. But a biochemical pathway is like an assembly line. Break one of the steps in the middle and the whole thing falls apart. That’s what glyphosate/round-up does.

This part of the story starts with an enzyme called 5-enolpyruvylshikimate-3-phosphate synthase (or EPSPS for short). Do you don’t have to understand what EPSPS does specifically**, what is important is that its job is an important step in making the three amino acids Tryptophan, Phenylalanine, and Tyrosine***.  When EPSPS can’t do its job, the next protein in the biochemical pathway won’t get the parts it needs to do its job, and in short order the whole pipeline shuts down, none of those three amino acids get produced, and the plant dies.

How does glyphosate keep EPSPS from doing it’s job? It imitates one of the the chemical building blocks EPSPS normally works with, so EPSPS proteins will bind to it like they would to the actual chemical compound. But since glyphosate isn’t the compound EPSPS actually work with, it sticks in the protein. If it helps you can think of this as feeding the wrong sort of paper into a printer, causing a paper jam. Lots of individual molecules of glyphosate get into each plant cell. They stick in EPSPS proteins floating within the cell, which keeps EPSPS from doing its job, and once EPSPS stops working, the plant cell can’t make the amino acids it needs to survive, and dies.

Glyphosate is very good at doing what it does: killing plants. And as weed-killers go, it’s a lot less nasty for animals since it works by breaking a protein animals don’t need or even have. But there is one problem. Some weeds are becoming less effected by the herbicide, able to survive larger and large doses.  There are a number of ways plants can evolve to survive large doses of glyphosate. Let’s talk about three:

1. The first, and probably most obvious, is to change the shape of the EPSPS protein so glyphosate can no longer jam the mechanism. As it turns out mutations that change which amino acid is used at one specific point can produce a version of the EPSPS gene that is less likely to be broken by glyphosate. Think of it as changing the design of a print so paper that would jam the mechanism either won’t fit in the printer at all or passes through harmlessly. This method of getting “superweed” powers has been used by malaysian goose-grass and and australian ryegrass.
2. A second way for plants to become superweeds is to stop transporting glyphosate around the plant. I don’t have a good printer metaphor for this one. Cells in the leaves of plants are mostly completely grown and don’t need to make as many new proteins as the rapidly dividing cells in meristems and newly developing leaves. When a farmer sprays glyphosate it will mostly land on the mature leaves of the plant. If plants can keep the herbicide in those leaves and keep it from traveling throughout the rest of the plants, they stand a better chance of survival, and that’s exactly what has been found in resistant stiffstalk rye and pigweed.
3. The first two methods are all well and good, but I probably wouldn’t have bother to write this post if it wasn’t for the method of resistance discovered in Amaranthus palmeri (one of the many species that share the common name pigweed). Palmer amaranth’s approach to resisting glyphosate is charming in its brute force. Resistant plants have duplicated the gene for EPSPS many times (up to 160 copies in some plants!). All those extra genes mean the plants produce a lot more EPSPS protein, so no matter how many individual EPSPSs get jammed by glyphosate molecules, there are still plenty more working EPSPSs to keep doing the job, and the biochemical pathway never stops. Sure a problem with paper jams can be fixed by more advanced printers, or more strict controls on what kind of paper is allowed into the building… but Palmer amaranth’s solution was simply to build a lot more printers.

Potentially there’s potentially a fourth way to develop glyphosate resistance, which would be for the resistant version of the EPSPS protein engineered into glyphosate resistant crops**** to be introgressed into wild relatives allowing those wild crop relatives to become herbicide resistant “super weeds”. This gets talked about a lot and clearly the risk is going to depend on a lot of factors*****. In researching this post I couldn’t find any papers describing herbicide resistant weeds that owe their resistance to a gene from an herbicide resistant crop. And given how much ink has been spilled on the subject, I would expect any such papers to makes a big splash.

*Biochemical pathways are just a bunch of steps needed to get from some molecule an organism already has, to some other molecule the organism wants. Usually each individual chemical change is performed by some specific protein, like workers on an assembly line. (Sometimes its even arranged like an assembly line with intermediate molecules being passed directly from one protein to another, although it isn’t always that way)

**Although if you’re interested you can read more about the details of the EPSPS protein here.

***The first two are certainly essential amino acids. Our bodies can produce our own tyrosine, but all we do is modify phenylalanine. We can’t make it from scratch.

****Weeds that resist glyphosate are “super weeds”, but I can’t imagine ever hearing the crops that resist the exact same herbicide called “super crops” .

*****How the crop reproduces, whether its being grown near any wild ancestors, how weedy those wild ancestors are to begin with, which crop alleles are in close linkage with the resistance gene (crop-like traits tend to make weeds much less successful).

Gaines, T., Zhang, W., Wang, D., Bukun, B., Chisholm, S., Shaner, D., Nissen, S., Patzoldt, W., Tranel, P., Culpepper, A., Grey, T., Webster, T., Vencill, W., Sammons, R., Jiang, J., Preston, C., Leach, J., & Westra, P. (2009). Gene amplification confers glyphosate resistance in Amaranthus palmeri Proceedings of the National Academy of Sciences, 107 (3), 1029-1034 DOI: 10.1073/pnas.0906649107

POWLES, S., & PRESTON, C. (2006). Evolved Glyphosate Resistance in Plants: Biochemical and Genetic Basis of Resistance Weed Technology, 20 (2), 282-289 DOI: 10.1614/WT-04-142R.1

Photo: ekpatterson, flickr (click photo to see in original context)

There are more differences in the genomes of two unrelated corn plants than between the genomes of a human and a chimpanzee (two species separated by 3.5 million years of evolution).

On the other hand, two unrelated human beings, members of the same species, have more than four times as many genetic differences as two unrelated heirloom tomatoes.

Genetic Diversity:

Corn vs. Corn > Human vs. Chimpanzee >> Human vs. Human >> Heirloom Tomato vs. Heirloom Tomato

Now the fact that any two human beings are more closely related to each other than either is to a chimpanzee should be obvious to anyone who gives it a moments thought.

I plan to poll my sections tomorrow to see how many of them would put corn and heirloom tomatoes in the opposite positions, but many have figured out my feelings about corn, so they’ll probably guess it’s a trap.

Libe slope in Ithaca, NY. Behind you are student dorms. At the top of the hill, campus starts. Photo: foreverdigital, flickr (click to see in original context)

When I was an undergraduate, there were exactly two sequenced plant genomes, rice and arabidopsis. And sure maybe I didn’t have to walk “ten miles to school, barefoot, in the snow, uphill, both ways”* the one way I did have to walk uphill (sometimes in the snow but always with shoes), was very uphill. But where was I?

Oh yeah, plant genome sequences. Kids getting into plant genomics these days don’t realize how easy they’ve got it. By my count (which may be low but I’m getting to that) there are ten published plant genomes, with several more unpublished genomes that are available in various states of completion, and lots more on the way.

Which brings me to what I was doing yesterday instead of writing an update for this website: trying to document the published plant genomes, the unpublished genomes that are available, and which new genomes we can expect to see published in the near future.

Please, if you find mistakes or know of additional flowering plant genomes I should mention, let me know! jcs98 (@) jamesandthegiantcorn.com.

If you don’t work in biology, it might be interesting to see which plants have sequenced genomes and how they’re related to each other.

*An explanation of this phrase.

Ears segregating for the colored aleurone mutant phenotype. Image courtesy of MG Neuffer via MaizeGDB.

Purple plant1's phenotype is highly variable depending on the genetic background the mutant is in. Images courtesy of MG Neuffer via MaizeGDB.

The two genes are also duplicates (homeologs) resulting from the maize whole genome duplication. From the picture below you can also see both the two genes and the regions they are in match up to single regions in rice and sorghum, two grasses that haven’t gone though a whole genome duplication since the great radiation of grass species that took place an estimated 50 million years ago (well after dinosaurs stopped walking the earth).

More interesting, at least to me, is the fact that there is NO gene equivalent to colored aleurone1 and purple plant1 in the region we’d expect to find such a gene in Brachypodium (the only other grass species with a sequenced genome)*. From all the genes that line up perfectly on either side we can predict the exact location the gene equivalent to colored aleurone1 and purple plant1 should be found in the Brachypodium genome. But the gene isn’t there…

The GEvo panel shown here can be regenerated at: http://tinyurl.com/yddlwor

The original version of this image had Pl1 mislabeled as C1. This version corrects that error.

For a couple more examples of comparisons between the four sequences grass genomes check out the Cogepedia post I spent this morning pulling together.

*The publication of which I celebrated just a little while ago.

I mention all this to explain why I was so excited to learn that her post this week sings the praises of a group of species near and dear to my heart, the grasses. The whole post is definitely worth a read. Even if you don’t learn something you didn’t already know, read it as a source of inspiration for telling OTHER people how cool grasses are. And the closing is truly excellent:

We usually talk of our domestication of grasses, and the ways in which we have evolved them: we have made plants with bigger, more nutritious seeds that don’t fall to the ground, for example.But their effect on us has been far more profound. Our domestication of grasses, 10,000 years ago or so, allowed the building of the first cities, and marks the start of civilization as we know it. Grasses thus enabled the flowering of a new kind of evolution, a kind not seen before in the history of life: the evolution of human culture.

Some of the comments are heart warming to read as well, although a bunch of people have fallen prey to the maize/corn confusion. (Explained in detail here)

*Speaking of cool science that most of the general public doesn’t know about: We’ve known for more than four years that mutations of the gene talpid2 in chickens cause chicken embyros to develop teeth, something we thought birds had lost the ability to do 60-80 million years ago (around the same time grass was bursting onto the world stage.) Don’t worry too much about getting bitten by a sabertoothed turkey, the toothed embryos have other problems that mean they don’t survive.

**There’s also a three-part video series based on the book that I can best describe as … odd.

* = tied for number of citations

** = some mutant alleles have kernel phenotypes.

If you want to become one of the famous mutant corn genes, it helps if you have an effect that is visible in corn kernels instead of only from fully grown plants.

And here is why:

• A geneticist could determine that the version of c1 that creates yellow kernels is recessive to the version that creates purple kernels just from looking at the ear of corn on left.
• Furthermore, they could tell you that both the male parent (the plant that provided the pollen) and the female parent (the plant on which the ear of corn grew) were both heteryzygous for the c1 genes (they each had one dominant version of the genes and one recessive version), and therefore the corn kernels the parent plants were grown from were both purple.
• They would know with certainty that all of the yellow kernels contain two recessive versions of the c1 gene.
• While they couldn’t predict with absolute certainty whether a specific purple corn kernel on that ear carried two dominant versions of the c1 gene or one dominant and one recessive version, they would know there was a 1/3 chance that kernel has two dominant copies, and a 2/3 chance it had one dominant and one recessive copy.
• That geneticist could make all sorts of predictions about what ears would look like in future generations depending on what colors of corn kernels were planted and which plants were mated with each other.

All this from a single picture of an ear of corn. For a phenotype seen in corn plants but not in kernels (like Knotted1), a geneticist would have to plant a row or more of corn seeds from an ear and examine the growing plants to get the same quantity of information.

And that is why mutations with kernel phenotypes have been so popular over a century of maize genetics research.

Corn Smut photo: oceandesetoiles, flickr (click to see photo in its original context)

And no that doesn’t mean corn pornography*. Corn smut, or Ustilago maydis, is a fungus that infects corn plants. It’s an old acquantance from my days working in the field. We always used to tell the new hires that corn smut was a rare delicacy in some countries (as we’d been told ourselves), but this was in the days before iPhones so until recently I never actually checked on this bit of received wisdom.

Turns out this particular bit of knowledge was true:

The immature galls, gathered two to three weeks after an ear of corn is infected, still retain moisture and, when cooked, have a flavor described as mushroom-like, sweet, savory, woody, and earthy.

More corn smut. Photo: moskatexugo, flickr (click to see photo in its original context)

I haven’t been able to figure out what the trade off in nutrition is between the ear of corn that is produced by a normal plant and the fungal galls that can be harvested from a plant infected with corn smut. I’d imagine corn smut provides more (and more complete) protein than an ear of corn (assuming corn smut is nutritionally similar to mushrooms.) But what’s the comparison in number of calories? The fungus is certainly sold at a higher price pound for pound.

My renewed interest in corn smut comes courtesy of a new paper** that came out in PLoS Biology describing how the fungus steals energy from infected corn plants without triggering the corn’s usual anti-fungal defenses. It’s an interesting read, you can check out the paper itself since PLoS Biology is open access, or Diane Kelley’s summary at “Science Made Cool.”

I’d seen a number of talks recently about another fungal parasite, powdery mildew in Arabidopsis, but somehow it’s much easier to focus on this stuff now that I can connect it back to corn. Even mammalian systems can be interesting*** once the make that connection.

*Please PLEASE don’t let that phrase start showing up in the search terms people use to find my site!

**Wahl R, Wippel K, Goos S, Kämper J, Sauer N (2010) A Novel High-Affinity Sucrose Transporter Is Required for Virulence of the Plant Pathogen Ustilago maydis. PLoS Biol 8(2): e1000303. doi:10.1371/journal.pbio.1000303

***The talk I’m practicing for Monday actually uses an example of a pheromone receptor in new world monkeys that was lost 23 million years ago in old world monkeys (including us humans).

But where was I? Oh yeah, gene counts. 32,690 high confidence genes*. Of those, how many have been studied individually?

While I don’t know that anyone knows the precise answer to that question, one indicator is how many maize genes were named before the maize genome was sequenced. People have been naming maize genes since before even the structure of DNA was known, based on the effect mutant version of the gene have on corn plants (for example: waxy1 or yellow stripe1). Later names might be based on the function of the gene (alcohol dehydrogenase1 or superoxide dismutase4), or anything else we know about the gene (wound induced protein1 or male flower specific18). The point being, if someone bothered to name a gene sometime during the last century of maize genetics, it was likely because they were studying it (to a greater or lesser extent). MaizeGDB keeps records of most of the named genes in maize and (excluding chloroplast and mitochondrial genes) I was able to find records of 1181 named genes in maize.

That’s less than 4% of the number of high confidence genes found within the maize genome, and at least a few of the named genes aren’t found within that group (see the first footnote for more details). Why is that number so low?

1. Each of the genes that has been studied in any detail probably represents some grad student’s doctoral thesis. While the tools have gotten better, the expectations for what is involved in characterizing a gene have risen too. I don’t have any statistics on how many maize genetics students earn their PhDs every year (and many of them will have worked on other kinds of projects than characterizing some new mutant gene), but it’s certainly not the thousands that would be required to characterize every gene in the genome in a short period of time.
2. Perhaps more importantly, the first genes to be studied are the ones with the best mutant phenotypes. To be a good mutant to study, breaking a gene should create something obviously different about the plant (it’s purple, or the tassel produces seeds like an ear instead of pollen, or the plants grow along the ground instead of standing upright), but not be so vital that embryos containing broken versions of the gene don’t develop at all. From a project that to knock out every gene in another plant Arabidopsis thaliana we know that many genes can be broken without any obvious effect on the plants that carry broken copies. That doesn’t mean there won’t be still be interesting things wrong with the plants when they’re studied in more detail, but such mutants were less likely to be identified early on. As for genes mutations are usually lethal, they can be studied (a friend in a lab downstairs is working with just such a mutant) but it certainly adds a whole new layer of difficulty to any research project so the genes better be involved in something interesting enough to justify the extra pain and suffering involved.

Now the situation isn’t nearly as grim as it might sound. Nature re-uses related genes over and over again both between and within species, so any time a researcher studies a new gene in detail, that information doesn’t just inform our knowledge of one particular gene in one particular species. Like a candle in a dark room, the information created by the study of a single gene will illuminate, to a greater or lesser extent, nearby genes (genes that have similar sequences to the gene being studied directly.) So even for a gene that’s never been studied in maize, we can make guesses about its function based on any related genes that have benefited from detailed study (either other genes in maize, in other grasses like barley or rice, other plants like arabidopsis or snapdragon, or even in animals or bacteria). While no geneticist worth their pollenating apron wouldn’t need experimental data before being CERTAIN of a gene’s function, knowing something about the functions of related genes is an excellent starting point.

I just finished some “free time” science looking at the classical genes of maize genetics (which displaced the time I normally spend writing for this site), so expect a couple more posts on related topics later this week.

*The good folks at maizesequence.org also produced a set of all the sequences they thought MIGHT be genes which, in addition to the filtered genes, includes ~70,000 more sequences that might or might not be genes. Many of these potential genes are computationally predicted, by programs that look at the underlying characteristics of the DNA sequence itself (how they work is outside my expertise and above my pay grade), but I can personally vouch for the fact that at least some of those “possible” maize genes are the real thing so the true number of genes contained within the maize genome is at least somewhat greater than the 32,690 reported with high confidence. This fact isn’t in any way a criticism of the people involved in sequencing and annotating the maize genome. The vast majority of the high confidence genes (called the filtered gene set) are real, and most of the other 70,000 genes (those included only in the working gene set (which also includes the genes from the filtered gene set)) are probably figments of a computer program’s imagination. Anywhere they chose to draw the line between the two groups was going to put some genes in the wrong category, and they did everything they could to minimize those miscategorizations.

**This doesn’t mean that the genes don’t have important jobs. You can imagine, for example, that genes involved in a plant’s ability to survive disease, water shortages, cold stress or heat stress all won’t create obvious problems for plants grown in the relatively pampered conditions we biologists try to provide for our research subjects when we aren’t actively studying what happens when we stress plants.

Keith Robinson writing over at Omics! Omics! posted Celebrating Citrus where he catelogs some of the diversity available to him from local grocery stores before pointing out a citrus review article that suggests all that diversity can be traced back to only three wild species and wraps it up by pointing out the project to sequence the sweet orange genome.

Imagine if you could have a whole series of clementine-like fruits, with the size & easy peeling characteristics but with the whole range of other citrus flavors and colors genetically grafted in — cara cara clementines and blood clementines and ruby red clementines and perhaps even sweet lemontines and key clemenlimes.

Highly recommended.

The Biogeography of Darwin’s Gourd is a post I discovered through research blogging (speaking of which I should really write another entry that meets their standards some day). The gourd of the title is Sicyos villosus, a cucurbit (the group of plants that includes squashes, melons, and pumpkins) collected by Darwin from one of the islands in the Galapagos the better part of two centuries ago … and never again recorded by science. At this point the dried sample collected by Darwin may be the only existence the species ever lived:

The analysis of the cucurbit’s DNA, extracted from the seed samples taken by Darwin, revealed that S. villosus is closest in relation to cucurbits in North America and Mexico. The species probably diverged roughly 4 mya, when the Galapagos were still geologically young. Dispersal was not human in origin, meaning long distance from the mainland, potentially from its spiny fruits stuck to birds, the authors suggest.

How cool is it that we can learn so much from a single sample of a species that has otherwise vanished from the earth?

Finally, by way of DailyKos, comes a pointer to this valentine’s day themed article, clearly written for the non-scientist, where a summary written by me seems superfluous given the title: Sex, Drugs, and Paleo-botany! And yes, the exclamation point is in the original title as well.

An anonymous indian tomato vendor in Chennai, Tamal Nadu. photo mckaysavage, flickr (click to see photo in it's original context)

First, since I didn’t explicitly state it in my previous post, the paper on the longer lasting tomatoes developed by India’s National Institute for Plant Genome Research didn’t report any data on how the RNAi knock-down tomatoes actually taste.* The tomatoes are nearly twice as firm as tomatoes in which these genes are NOT knocked down, so it’s possible they’d seem unpleasantly crunchy, I don’t know how doubling the firmness of a tomato translates into the feeling when a person bites into one.

On the other hand, if the tomatoes do turn out to be tasty and delicious, it’s quite possible the trait could be replicated without genetic engineering. And if that turns out to be true, it’s absolutely the approach anyone developing longer lasting farmers to Indian farmers, or farmers anywhere, should take (for why I’m saying this, check out the bit in bold further into this post). The synthetic microRNAs used in their experiments reduced gene expression by at least 99%, so, if it turns out that remained <1% isn’t playing a key role, the researchers at NIPGR have effectively created knock out lines for each of the two genes they were studying. Knocked out genes (genes so broken they don’t work anymore) has been a key part of genetics since before the word gene even existed. (Mendel in the 1850s and 1860s and Wilhelm Johannsen in 1905 respectively) With a couple of known targets, and a target phenotype that’s known to be worth the effort, creating tomatoes with “naturally” broken copies of the gene is possible and probably worth the effort to avoid the expense and controversy associated with trying to commercialize a new genetically engineered trait.

The mutant (left) and wildtype (right) tomatoes from mutant line e2615m1 from the mutant population cited below. Photo from the searchable database of identified mutant phenotypes at: http://zamir.sgn.cornell.edu/mutants/

Back in 2004 a paper** from two research groups in Israel described a saturation mutagenesis population*** of tomatoes created using EMS**** and fast neutron***** techniques. Screening 13,000 inbred lines for knock outs of either of these two genes would take a fair bit of time and money, but less than is involved trying to get approval of a genetically engineered trait (especially in India, where a political battle over their first genetically engineered food crop, an insect resistant breed of eggplant is still ongoing).

If it weren’t for (what I consider to be) irrational fears about genetic engineering and actions of people who exploit those fears, the arguments of speed and cost would instead rest with the genetically engineered RNAi knock downs already created in this study. Given the world we live in, and given there is an way to get the same benefits without genetic engineering in this particular case, getting the benefits of cheaper produce to people who could use the vitamins, and higher effective yields to farmers who could use the money must take priority. In the mean time people like you and I will just have to keep doing our best to combat that ignorance and fear so someday the deciding factor will be whatever technique is safest, fastest, and makes the most efficient use of scarce resources, not what people have, apparently arbitrarily, decided to natural or unnatural.

And as I said above, we don’t even know if the tomatoes are tasty, or if the NIPGR is or will be working on creating varieties of tomatoes with this trait for use by India’s farmers in the first place, so speculation on this paper may have gotten a bit too far ahead of itself.

*If they people who worked on the project are at all after my own heart, I’m sure they’ve tried the tomatoes for themselves, but subjective judgements like taste aren’t going to make it into a PNAS paper on fruit ripening (and its possible consuming genetically engineered tomatoes that haven’t been approved would technically be breaking the law in India, in which case the researchers would even less inclined to publicize any off the books tasting they did on their own.)

**Menda, N et al “In Silico Screening of a Saturated Mutation Library of Tomato” The Plant Journal DOI: 10.1111/j.1365-313X.2004.02088.x

***A mutagenesis population is a group of plant lines that have all been exposed so some mutating agent (see the footnotes on EMS and fast neutron). A saturation mutagenesis population is one where,  based on the number of lines of plants in the population and an estimate of the number of mutated genes in each line, a mutation of (almost) any gene in the genome will likely be found somewhere in the population. The exception being really genes that are so bad to lose that plants carry even one broken copy, or gene and pollen cells (which only have one copy to begin with), die without being able to reproduce.

****EMS stands for Ethyl methanesulfonate, a chemical that creates a specific kind of mutation in the genome of a plant (changing some Gs into Ts). Exposing plants to specific doses of this chemical creates (on average) predictable numbers of mutations in the genome.

*****Fast neutron mutagenesis is less common than EMS, likely because creating fast neutrons involves the use of radioactive substances (one source I found specifically cited uranium aluminum alloys). The advantage of fast neutron mutagenesis is that as the neutrons tear through cells they tend to rip away large chunks of DNA (hundreds or thousands of As, Cs, Ts, and Gs at once) which makes the resulting mutants more likely to completely lose the function of a gene (imagine using EMS to create mutations as changing a couple of key letters in a recipe, and using fast neutrons as tearing out a whole page of a cook book and throwing it away).