Transgenic Sweet-Potatoes

One of the first tools available to biologists seeking to make transgenic plants was a natural bacteria called Agrobacterium. The bacteria infects plants and induces the production of auxins and cytokinins, plant hormones that together trigger the growth of a disordered plant tissue called a gall. The gall provides nutrients that the bacteria then uses for its own growth. The bacteria pulls off this trick by inserting genetic information into plant cells. It can do this because it carries a plasmid that can function both in its own cells and in the cells of a plant. When the bacteria infects the plant, the plasmid is transferred into the plant's cells. Once there, the plasmid integrates into the genome of the plant and activates. The bacteria are natural genetic engineers.

Genetic remnants can be left behind whenever the bacteria does this trick and nowadays we can do genome sequencing to find such remnants. Kyndt et al found remnants of this process in sweet-potatoes (Ipomea batatas). In particular, they found such a fragment inserted into an intron of an F-box gene in the sweet-potato genome. Interestingly, this interruption is found in all domesticated sweet-potatoes they examined and not in wild related species.

The insertion contains multiple complete genes and results in the interruption of the F-box gene. F-box genes are generally involved in protein degradation and in plants they're involved in the regulation of development during growth. It isn't clear that the insertion they found results in a change in the development of the plant associated with domestication, however. They are still working on this.

Oh yes, did I mention they found this fragment in all samples they examined of domesticated sweet-potatoes? This means that every single sweet-potato is a transgenic organism, a GMO. We didn't make it a GMO, but there is no way around it, every sweet-potato is a GMO.

Every sweet-potato is a GMO.

Are you against GMOs? Then don't eat any more sweet-potatoes. Are you actually against the business practices of Monsanto surrounding their GMO and herbicide products? Then rail against those business practices instead of the red herring that GMOs are in the overall story.



References:

1. Agrobacterium: en.wikipedia.org/wiki/Agrobacterium
2. Kyndt et al, 2015: m.pnas.org/content/early/2015/04/14/1419685112.full.pdf
3. F-box genes: en.wikipedia.org/wiki/F-box_protein
4. Red herring: en.wikipedia.org/wiki/Red_herring

Glucose Metabolism (Burying the Lede)

1. Glycolysis

The process of glycolysis (Fig 1) breaks down a single 6-carbon glucose molecule into two 3-carbon pyruvate molecules. Glycolysis is the central metabolic pathway for converting sugars into energy for all known living things. Two ATP and two NADH are generated for each glucose molecule processed.

2.  A. Krebs Cycle.  B. Carbons.

If oxygen is available, the resulting pyruvate can then be processed by the Krebs cycle (Fig 2A) to produce two more ATP, as well as eight NADH and two FADH₂. The three carbons from each new pyruvate molecule take more than two complete cycles of the Krebs Cycle to be converted into CO₂ (Fig 2B).

The NADH and FADH₂ can be used in other pathways or they can be used to generate further ATP through the process of oxidative phosphorylation in the mitochondria. Three ATP can be generated from each NADH and two ATP can be generated from each FADH₂. The three processes together generate 38 ATP from each glucose molecule metabolized.

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3. Fermentation pathways.

When oxygen isn't available, alternate fermentation pathways (Fig 3) are used to get rid of the excess pyruvate that builds up from glycolysis. Because the NADH produced in glycolysis is consumed during ethanol and lactic acid fermentation (fig 3A and 3C), only two ATP are generated in these two fermentation pathways for each glucose molecule metabolized. In acetate fermentation (fig 3B), four ATPs and two NADHs are produced for a total of 10 ATP equivalents.

The net result is that oxidative metabolism can generate 38 ATPs compared to the 2 ATPs of ethanol and lactic acid fermentation and the 10 ATPs in acetate fermentation, for each glucose metabolized. Oxidative metabolism is far more efficient at generating biological energy in the form of ATP.

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In the fermentation pathway leading to ethanol, one-third of the carbons are lost as CO₂. If a pathway could be engineered to capture that CO₂ and incorporate it into another ethanol, the efficiency of the process for creating ethanol could be increased by 50%. This would mean more fuel could be produced more cheaply and ethanol-based fuels would become more cost-effective overall.

4.  A. NOG & EtOH fermentation.  B. Carbon rearrangement.

This is exactly what Igor W. Bogorad, Tzu-Shyang Lin, and James C. Liao argue they have accomplished in what they call non-oxidative glycolysis (NOG). I didn't like their figures because they didn't illustrate the pathways taken by individual carbons or illustrate the final energy balance, so I made my own figure for the first pathway they describe.

In figure 4B, three erythrose 4-phosphate (E4P) molecules are rearranged to make two fructose 6-phosphate molecules (F6P). Each initial E4P molecule is colored distinctly (magenta, red, and blue) and then the carbons in later steps are colored to indicate which E4P they came from. In the end, one F6P is built from three carbons from each of two E4Ps while the other F6P is built from all four carbons of one E4P and the remaining one carbon each from the first two E4Ps.

NOG is able to convert a single glucose molecule into three acetyl-CoA molecules, with no net change in ATP levels or loss of carbon in the form of CO₂. Each acetyl-CoA molecule converting to a molecule of ethanol, however, absorbs two NADH molecules (equivalent to 6 ATPs). The final results for this pathway are the consumption of the equivalent of 18 ATPs (3 for each NADH consumed) for each glucose molecule converted to ethanol. This means that the cell would have to process 9 glucose molecules by typical glycolysis paired with anaerobic fermentation for each 1 glucose molecule processed by this version of NOG to maintain a zero energy balance. This isn't a biologically efficient process at all.

5. NOG and acetate fermentation.

They mention integrating this pathway with carbon assimilation pathways to convert CO₂ into fuel. Because the ethanolic fermentation pathway after NOG is so energetically unfavorable, I'm not sure how this would work. I'll have to later examine the energetics of the pathway when tied to carbon assimilation pathways to see if the argument actually makes sense.

If instead the acetyl-CoA is fermented to acetate or to acetic acid as a terminal product, there would be a net gain of [3][2] ATPs per glucose. This final version is discussed in short form in Bogorad's paper and is energetically useful, [producing 50% more ATP than the typical glycolysis linked to ethanol or lactic acid fermentation, though still] [producing the same amount of ATP as typical glyclolysis linked to ethanolic or lactic acid fermentation. This is] well below the 10 ATPs per glucose produced through typical glycolysis linked to acetate or acetic acid fermentation.

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5. NOG & Krebs Cycle.

What happens if we process the resulting acetyl-CoA by the Krebs Cycle instead of anaerobic fermentation?

The NOG pathway generates 0 ATP compared to the 8 ATP generated during glycolysis. However, the NOG pathway generates 3 acetyl-CoA compared to the 2 acetyl-CoA generated during glycolysis.

Each acetyl-CoA generates the equivalent of 15 ATPs through the Krebs Cycle so the combination of NOG and the Krebs Cycle will generate a total of 45 ATPs for each glucose molecule processed. This is more than the 38 ATPs generated by the combination of glycolysis and the Krebs Cycle.

This pathway produces 25% more biologically usable energy per glucose molecule than what our biology uses, with precisely the same chemical inputs and outputs. This is an amazing result that should have been the headline result of Bogorad's research paper. It would have been the first thing I talked about in this blog post, except that I needed to build the story that provides context for the significance of this result.

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NOG-acetic-fermentation is more productive than glycolysis-lactic/ethanolic-fermentation and NOG-Krebs-cycle is more productive than glycolysis-Krebs-cycle in terms of ATP output per glucose metabolized.

Why does our biology use a relatively inefficient method of glycolysis?

Once glycolysis evolved, other metabolic pathways would have evolved to take advantage of the various intermediates of glycolysis. This would have the effect of making dramatic changes to the pathway more costly, locking the pathway into its present form. This appears to be the argument preferred by the authors.

Another possibility is that the NOG pathway was simply too large of a transition from the existing glycolytic pathway to have evolved naturally. Acetic fermentation isn't a common pathway even though it produces far more ATP energy than the common ethanol and lactic acid fermentation pathways. Because acetic fermentation is a prerequisite to NOG being energetically productive, there have been relatively few possibilities for NOG to have evolved naturally.

That pathways with higher efficiency exist but haven't become common is suggestive of limits on how living things can naturally evolve and that other considerations than efficiency at producing ATP are significant.


References:

1. Non-oxidative glycolysis: www.nature.com/nature/journal/v502/n7473/full/nature12575.html
• NOG figures: www.nature.com/nature/journal/v502/n7473/fig_tab/nature12575_ft.html
2. Alternate source for paper: www.bnl.gov/biosciences/JournalClubDocuments/YuanhengCai-Reprint.pdf
3. Acetate fermentation: www.vet.ed.ac.uk/clive/cal/rumencal/frames/frmfibro.html
4. Acetic Acid fermentation: www.pnas.org/content/105/37/13769.figures-only 
5. Krebs cycle: en.wikipedia.org/wiki/Citric_acid_cycle
6. Ethanol fermentation: en.wikipedia.org/wiki/Ethanol_fermentation
7. Lactic acid fermentation: en.wikipedia.org/wiki/Lactic_acid_fermentation

Other pathways of interest:

1. Entner-Doudoroff pathway: en.wikipedia.org/wiki/Entner–Doudoroff_pathway
2. Pentose Phosphate pathway: en.wikipedia.org/wiki/Pentose_phosphate_pathway

Biological Diversity

In recent conversations I've had with biologists at a plant breeding symposium at UMN and other local events, I've come across people using the term "diversity" in rather distinctively different ways.

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A researcher was described how low diversity soybeans were, as an argument for the fast-neutron mutagenesis project he was working on to develop new useful diversity. I didn't believe soybeans to be a low diversity crop, as there are so many wild and weedy forms available in the center of diversity for the species in China. He then made some comment about the limited diversity brought to the United States from Asia...  which made me wonder why he would limit himself to what is locally available, when strains from the whole world are available with some effort.

The closing keynote speaker was later discussing his 34 year career of soybean breeding and started off by describing the great diversity available in soybeans for breeders to work with. I then realized that the first researcher and I had been using different measures of diversity. He was using "diversity" to mean what was commonly available locally (definition #1 below), while I (and the keynote speaker) were using "diversity" to mean the range of genotypes available worldwide (definition #2 below).

Population genotype structures:
Local: AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAABB
World: AAAAAAAAAAAAAAAAAAAAAAAAAAAAABBCDEFGHIJKLMN

1. Range of genotypes locally available. The population approximates just genotype A, so under this definition the crop species has a very low diversity.

2. Range of genotypes in a species. The population includes 14 distinct genotypes (ABCDEFGHIJKLMN), so under this definition the crop species has a very high diversity.

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At another event, a speaker was talking about how some regions of the genome for his study plant species had very low diversity and that this indicated recent introgression from a related species because the rest of the genome had a very high diversity.

I commented that I would have to look into the math behind it to better grasp what he was talking about.  He shrugged off the need for this and said the region was determined as having low diversity using a hidden-markov-model (HMM) approach.

I pondered on his statement. The HMM approach is used to identify transitions between states as you move along the axis of a dataset. In the case of the speaker's research, the HMM would calculate the most probably coordinates in the genome for transitions from high diversity to low diversity and back. Unfortunately, this still doesn't explain what he meant by "diversity". As the speaker was talking about a region of the genome having lower diversity than average for the species(individual?), there are two ways I can interpret his use of "diversity".

3. The amount of heterozygosity within an individual. A highly inbred individual will have a very low "diversity", while a highly outbred hybrid individual will have a very high "diversity".

4. The variation in haplotypes across a population. Haplotypes are distinct coordinately traveling regions of genetic information. The more haplotypes in a region, the more diverse the population is. A You would need to sequence a relatively large number of individuals to get a glimpse at the sort of data this analysis would require.

I have the feeling he was using "diversity" to mean a change in some measure across the genome of an individual (definition #3 above), rather than a measure of the population of the species (definition #4 above).

If he meant "diversity" like definition #3 above, then I would interpret a region of the genome with much lower level of heterozygosity to indicate a recent loss-of-heterozygosity (LOH) event, such as a damaged chromosome region being repaired by replacement with sequence from the intact other homolog to the chromosome. If he meant "diversity" like definition #4 above, then I would interpret such a region to indicate a recent selective sweep. In neither case does the data suggest to me there has been an introgression from an unspecified near relative with a higher propensity to self.

I really wish I had been able to get more clarity from the speaker about what he meant. I also wish he hadn't dismissed my interest in his project so quickly.

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The meaning of even commonly used words can drift between different groups of speakers. In science it is very important to be clear in what you mean by the terms you use, even for the words that don't seem to be jargon.


References:

1. HMM: en.wikipedia.org/wiki/Hidden_Markov_model
2. Haplotypes: en.wikipedia.org/wiki/Haplotype

Astrobiology : The basics of life.

Life as we know it is composed of three basic types of chemical compounds: nucleic acids (DNA & RNA), proteins, and lipids. One aspect of the study of abiogenesis (the origin of life) has been to determine how the basic compounds could have been formed from simple chemical precursors that we already know can be formed from simple physical and chemical processes.

One of the first experiments in to examine how the basic biomolecules could have been formed was performed in 1952 and is known as the Miller-Urey experiment. In this experiment, a selection of simple reducing gases was repeatedly condensed and boiled while being exposed to electric sparks. The experiment was setup to mimic the conditions of the early atmosphere here on Earth. The reducing gases are those that would have been generated from geologic processes before living things started adding oxygen to the mix. The sparks represented lightening, which was thought of as a plausible energy source that could drive the reactions to generate interesting chemistry.

The experiment was able to produce hydrogen cyanide (HCN), formaldehyde (CH₂O), and other simple molecules. These molecules then reacted to form over 40 different amino acids, in sufficient amounts to color the condensed liquid pink after a day. Several variations of the experiment have been done that produce different mixes of small biomolecules, including all the purines and pyrimidines used in the bases of RNA/DNA as well as the ribose sugars used to construct nucleotides.

Most of these experiments required the presence of ammonia (NH₄) and methane (CH₃) to produce interesting biomolecules. This is a problem, because these gases are now thought to be relatively rare in the early atmosphere because of their absence in the volcanic gas emissions that would have contributed heavily to the early atmosphere.

A new set of conditions has been identified (Patel, Nature 2015) that is able to generate the precursors to amino acids, nucleic acids (sugars and purines/pyrimidines), and lipids. The key components were hydrogen cyanide (HCN) and hydrogen sulfide (HS) from the atmosphere and ultraviolet light from the sun interacting in a water (H₂O) bath. The process was sped up by the presence of dissolved copper ions. Interestingly, the proposed reactions generate all the basic compounds used in our type of life, but also don't generate many other simple compounds that our type of life doesn't use.

I consider this topic to be part of astrobiology because the same basic physical processes which are studied in research into abiogenesis here on Earth have also been playing out at sites throughout the universe. The same basic compounds of life have been generated on wet worlds wherever they are found. This not only suggests that life may be ubiquitous through the universe, but that when life is found on planets like ours that it may also be composed of similar basic types of molecules.

Lots of other simple chemicals have been identified in space using spectroscopy. These have also been generated through common physical and chemical processes and may represent the basic chemicals found in other types of life that started in environments distinct from our own.


References

1. Miller-Urey experiment: en.wikipedia.org/wiki/Miller%E2%80%93Urey_experiment
2. Recent analysis of Miller-Urey: www.ncbi.nlm.nih.gov/pubmed/23340907
3. Patel, Nature 2015: http://www.nature.com/nchem/journal/v7/n4/full/nchem.2202.html
4. Molecules in space: en.wikipedia.org/wiki/List_of_interstellar_and_circumstellar_molecules

Convergence on the Seaside

Cakile maritima flowers.

I found this plant growing on the beach on a trip to southern California two years ago. The four-petaled flower and shape of the seed pods told me it was in the family Brassicacea, like the wild radishes which are common in the region. The thick and succulent leaves, however, indicated this plant was unlike any plant in the family I had seen before. I collected a few seed pods, hoping to later identify the plant.

After periodic internet searches over the last two years, whenever I found myself thinking about the plant, I finally found an image of a flower with the right shape and color that also grows in beach habitats above the high-water line. I've identified the plant as Cakile maritima (European Sea-Rocket). It and the related C. edentula (American Sea-Rocket) and C. lanceolata (Coastal Sea-Rocket) have small seed pods which contain one or two seeds and are dispersed by floating in water. The pods and leaves differ in shape between the species, but they are otherwise very similar.

C. maritima seed pods.

The species in this genus are generally described as edible, with leaves tasting spicy like horseradish. The plant I found had leaves which tasted mostly sweet. This may be just because I chose young leaves to taste, or I might have lucked onto a plant that was mild instead of hot. Hopefully the seeds I've stored will germinate so I can find out. Either way, I see it as an interesting plant to develop for the salad garden.

Sea-Rocket is commonly described as edible, but it doesn't seem it was ever a major crop. I've found one reference to it being grown in a garden in 1596-1599, but it isn't clear if was grown for vegetable or botanical use.

Another interesting beach-side plant in the Brassicaceae is Crambe maritima (Sea-Kale). Both plants have succulent leaves and dry fruit that effectively float and disperse their seeds. I find interesting that the two species evolved convergently from related ancestors to similar forms in the shared environment of European coastlines.

Sea-Kale appears to have been commercially harvested in Roman times and was cultivated in Europe from around the 1600s, but went out of fashion around the time of WWII.

References:

• Cakile maritima (Sea-Rocket)
• en.wikipedia.org/wiki/Cakile_maritima
• Biology of: onlinelibrary.wiley.com/doi/10.1111/j.1365-2745.2006.01131.x/full
• 1596-1599 garden: books.google.com/books?id=nmwZAAAAYAAJ&pg=PA28#v=onepage 
• Crambe maritima (Sea-Kale)
• en.wikipedia.org/wiki/Crambe_maritima
• edible: www.eattheweeds.com/sea-kale/

Biology in the Snow

All through this deep Minnesota winter, I've been trying to think of some observations or discussion I could have about the biology of snow.

There are lots of interesting little things to learn about how living things deal with snow. Some plants grow roots into snow-banks to extract nitrogen before it is delivered to other plants in melt-water. Various bugs are specialized to deal with the cold and are active in and on snow. There are plants that heat themselves up in spring, melting away snow, so their flowers can get pollinated. I find these and many other things to be very interesting...  but it is difficult to go out and examine biology in the snow so I can have some direct observations to talk about.

The one exception is the observation of tracks in new snow. The photo at left shows the track left from a mouse scampering from near my front door (at right) to the top of my deck stairway (at left). As it bounded along, its larger hind feet landed ahead of the smaller front feet. This track doesn't tell a great deal, but it does highlight the amazing energetic feat of a tiny little rodent running around in the cold snow instead of freezing into a solid little mouse-cube.

You can learn a lot about what the animals are doing by reading their tracks.

A persistent mouse track-highway has revealed a well-used path between a rock-pile shelter and the base of a near-by blue spruce, where the mice are presumably foraging.

Other tracks have revealed the under-snow explorations of short-tailed voles. Many of these tracks become very obvious when the snow begins to melt. Long arcing paths are left as they forage for seeds and other tasty plant bits.

One time, I found tracks from a mouse bounding along the top of the snow crossing over the tracks left by a burrowing vole. The two rodents have very distinct methods of getting around in the snow. Mice hurry along from shelter to shelter, while voles tunnel along under the snow's surface. Both strategies minimize their exposure to cold wind.

Recently, the tracks of two coyotes revealed how they sauntered through the yard while one playfully ran around the other. I knew coyotes were in the neighborhood because we saw one crossing the road a few miles away, but I had never seen or heard them in the yard.

I've found the unique impression formed when an owl or crow lands heavily in the snow while trying to capture a burrowing vole.

Unfortunately, tracks in the snow can be very hard to photograph. You're trying to capture a white impression in white snow. The tracks are plain to see in 3D, but the 2D images from a camera are usually unclear. The mouse-track image above took several tries and then further post-processing to highlight the tracks that were immediately visible in person. Even after that work, I'm still pretty sure that the photo only turned out because the footprints were clearing away the snow to reveal the darker colored decking material beneath.

So, until I can figure out a good way to capture the images for presentation and discussion, all I can do is suggest you pay attention to the tracks being left all around you in the snow.

References:

• Snow-roots: www.academia.edu/2517365/New_nitrogen_uptake_strategy_specialized_snow_roots
• Snow-bugs: www.microscopy-uk.org.uk/mag/indexmag.html?http://www.microscopy-uk.org.uk/mag/artjan99/basnow.html
• Skunk Cabbage: www.damninteresting.com/warm-blooded-plants/

Publish or Perish

Most of my biology-focused posts have been about topics related to garden projects I've done or have been thinking about. The subject of gardening does account for much of the biology I spend my free-time thinking about, especially now in the middle of a winter-bound Minnesota. It is also far easier to examine the growth and interactions of living things in the garden than it is to go on expeditions or develop lab protocols to explore the diversity of biological topics.

I've avoided talking in detail about the biology I do at work because the work is on-going and to publicize it here might detract from the process of publishing the work in more formal contexts once the projects reach usable conclusions. Sometimes a key result can be quickly replicated once the idea behind it has been developed, so talking about results too early can be asking for some competitor to beat you to publication. I've defended my thesis and dealt with graduate school bureaucracy sufficiently to be awarded my PhD. I can now refer to myself as "Darren Abbey, PhD" in professional contexts. (I can also refer to myself as "The Doctor" (Dr. Who?) in certain social contexts.)

Along the way, I've written or contributed to several research publications. The following list is the publications I've been received name credit for my contributions, from oldest to most recent.

1. Gale CA, Leonard MD, Finley KR, Christensen L, McClellan M, Abbey D, Kurischko C, Bensen E, Tzafrir I, Kauffman S, Becker J, Berman J. (2009) SLA2 mutations cause SWE1-mediated cell cycle phenotypes in Candida albicans and Saccharomyces cerevisiae. Microbiology. 155(Pt 12):3847-59. [PMID: 19778960]
    
2. Forche A, Abbey D, Pisithkul T, Weinzierl MA, Ringstrom T, Bruck D, Petersen K, Berman J. (2011) Stress alters rates and types of loss of heterozygosity in Candida albicans. MBio. 2(4). [PMID: 21791579]
    
3. Abbey D, Hickman M, Gresham D, Berman J. (2012) High-Resolution SNP/CGH Microarrays Reveal the Accumulation of Loss of Heterozygosity in Commonly Used Candida albicans Strains. G3 (Bethesda). 1(7):523-30. Erratum in: G3 (Bethesda). 2(11):1473. [PMID: 22384363]
    
4. Hickman MA, Zeng G, Forche A, Hirakawa MP, Abbey D, Harrison BD, Wang YM, Su CH, Bennett RJ, Wang Y, Berman J. (2013) The 'obligate diploid' Candida albicans forms mating-competent haploids. Nature. 494(7435):55-9. [PMID: 23364695]
    
5. Abbey DA, Funt J, Lurie-Weinberger MN, Thompson DA, Regev A, Myers CL, Berman J. (2014) YMAP: a pipeline for visualization of copy number variation and loss of heterozygosity in eukaryotic pathogens. Genome Med. 6(11):100. [PMID: 25505934]
    
6. Ford CB, Funt JM, Abbey D, Issi L, Guiducci C, Martinez DA, Delorey T, Li BY, White TC, Cuomo C, Rao RP, Berman J, Thompson DA, Regev A. (2015) The evolution of drug resistance in clinical isolates of Candida albicans. Elife. 4. [PMID: 25646566]
    

I've also got a couple others in the pipeline. For one I'm looking for a target journal, for the other I'm still deciding exactly how to present the results. I'll let you know more details once they're further along the way to publication.

The projects a grad student works on depends on a mix of the lab they end up in and their personal style of problem solving. I ended up in a Candida albicans lab and brought to it a heavy computational approach. The mix between the two is the realm of computational biology, the topic I find myself most connected to.

The academic life can readily be described as, "Publish or Perish". With six name papers from my time in grad school, I've done alright. Now I just have to figure out the next step.