Category Archives: basics

On becoming a tree…

“As long as you keep getting born, it’s all right to die sometimes”

Orson Scott Card, The Speaker for the Dead.

I’ve written many times about the differences between autotrophs and heterotrophs on this blog. Loyal readers will know I have a hard-line stance that humans fall decidedly into the heterotroph camp. Nevertheless, some art projects blur the lines between person and plant. So I was more than a little intrigued when a link for the Bios Urn came across my Facebook feed.

BiosUrn

The product offers an alternative to the traditional cemetery as your eternal destination. It’s a special biodegradable urn in which your ashes (or a loved one’s or a pet’s ashes) can be placed along with a tree seed. The whole thing is planted in the ground at a cemetery or other special location. The recycled carbon atoms of your body become the growth medium for a tree that will grow and live on after your death.

biosurn2

It’s beautiful. I get it and as someone who often quotes the Lorax, I’m all for any excuse to plant trees. But honestly, the first thing it made me think of was my all-time favorite book The Speaker for the Dead by Orson Scott Card.* Within the world of this book, humans live on a colony planet that happens to have another species of sentient beings called the Pequeninos. These organisms spend the majority of their life cycle as mammal-like beings, but can pass on into a third life as a tree provided they have been vivisected. It’s pretty gruesome and obviously humans don’t work this way, so cultural misunderstandings ensue.

However, because I’m a stickler for scientific accuracy when it comes to plant science (Hey, someone’s gotta be.), I have one problem with the overly simplistic marketing scheme of the Bios Urn. You don’t actually become the tree. For those of you saying, “I know, I know I won’t be a tree, but in the circle of life my molecules will become part of this tree.” I’ll still be over here at my blog shaking my head, “No, that’s not really how that works.” Here’s a reminder of the photosynthetic equation:

6 CO2 + 6 H2O (+ light) → C6H12O6 + 6 O2

Plants accumulate carbon and mass from atmospheric CO2, not carbon ash or even organic carbon compounds in the soil. The truth is that if you planted your ashes with your tree seed, your carbon would still remain locked in the soil for many years until eventually it is metabolized by microorganisms in the soil and released as CO2 as part of their respiration. This is not a very quick way to turn over your carbon molecules, and your molecules have millennia before they ever become a part of any tree. If you truly wanted your carbon molecules to incorporate into some kind of plant matter, then you need to find a way to carbonate your deceased body. Human carbonated essence could be stored in canisters and then used to supply CO2 to your plant of choice. You could be part of that camellia bush in your front yard, your favorite LSU Oak tree, the General Sherman etc. Scientifically accurate, but good luck marketing that compared to cremation.**

All of this highlights a common misconception about photosynthesis- it’s hard to comprehend how mass can accumulate into living things from air. Our gut tells us mass must come from something more substantial. We memorize the photosynthetic equation in elementary school, but few of us grasp its consequences and really believe it.

“This is how humans are: We question all our beliefs, except for the ones that we really believe in, and those we never think to question.”

Orson Scott Card, The Speaker for the Dead

So, when you die, ask someone to plant a tree or something in your memory. Do it for someone else you’d like to remember, but ashes not really required. Or buy a BiosUrn or put the ashes in a degradable coir starter pot from your local garden center, but you’ll probably want to supplement them with some form of NPK fertilizer. It will help the seed more than your carbon atoms.

Johnna

*It’s a sequel to Ender’s Game for those of you that may not be SciFi freaks.

**Although, in-home soda fountains are a thing now, so if there is a way to carbonate deceased relatives and store them in the handy canisters… well, you get the idea.

Links and References:

https://urnabios.com/

http://en.wikipedia.org/wiki/Speaker_for_the_Dead

The Molecular Biology Code

“You’re pirates. Hang the code. Hang the rules. They’re more like guidelines anyway.”              Elizabeth, The Pirates of the Caribbean: The Curse of the Black Pearl

pirate

Molecular biology, the overall practice of manipulating DNA sequences using biological scissors and glue, is a major component of any modern research lab. Its techniques have been revolutionary with respect to expanding the types of questions scientists can answer. While it may be grounded in the fundamentals of biology’s central dogma, molecular biology is often practiced with rituals and superstitions characteristic of pirates and other Caribbean stereotypes. And, like the pirate code, molecular biology contains as many exceptions as rules. What one molecular biologist may swear by is probably not so strictly enforced by another. After all, with most molecular biology construction projects, you only need it to work once and hope the sequence is right. So what I write here today are more along the lines of guidelines for cloning success.

  1. The enzyme is the last thing to add to any reaction.
  2. Do not vortex enzyme reactions.
  3. Use a new tip for your pipettor each time.
  4. Prepare a master mix for multiple reactions.
  5. Avoid gel purification of DNA fragments if at all possible.
  6. I’m not convinced that the enzyme ligase does anything, but add it anyway.
  7. Use restriction enzymes until you have evidence they don’t work anymore.
  8. Do the controls.
  9. Phosphatase treat your vector or insert but not both.
  10. Selection > Screen > Hope
  11. Sequence your finished construct to verify it is error free.
  12. Make freezer stocks.

See below for more explanations…

The enzyme is the last thing to add to any reaction.

This is basically another way of using Biochemistry Rule #4 (Use a buffer). Your expensive enzyme will likely be ruined if you put it in a tube of water or other dilute solution with no buffering capacity. So, pipet everything else first (DNA, nucleotides, water, concentrated buffer solution etc) and add the enzyme last. I would even argue that you shouldn’t even take the enzyme out of the freezer until you are ready to pipet it. Always keep it on ice or a cold block container. NEVER let it sit around on your lab bench at room temperature. If you were thinking of taking your reactions right away to the necessary water bath or instrument, think again. PUT THE ENZYME BACK IN THE FREEZER FIRST!!! That way, there’s no chance of you moving onto the next experiment without your precious enzyme safely stored away. Now, whether or not you choose to dilute your reactions with Holy Water or otherwise blessed dH2O is up to the individual scientist.

Do not vortex enzyme reactions.

After you’ve taken the care to ensure your enzyme is gently pipetted into an appropriately buffered reaction, don’t kill it by mechanically ruining it. You will need to physically mix the enzyme into the reaction because the enzyme exists in a glycerol solution that will sink to the bottom of your reaction. Do this by gently pipetting up and down or flicking the tube with your fingers. You may develop your own distinct style of mixing akin to a secret handshake. This routine is usually the superstitious result of a single instance that ‘the experiment worked when I did it this way.’

Use a new tip for your pipettor each time.

Reagents are precious and must not be contaminated. Use a fresh tip every time for each reagent and sample. Contaminated reagents may not ruin the experiment you are doing today, but they will ruin yours tomorrow and all of your labmates’ experiments. Be mindful when pipetting in general. If you can’t remember whether you added a reagent or not, just start over. Soon enough you will have your own Rain Man-like process for pipetting your reactions. On top of this, you may also develop your own style for using tips out of the box. Some of you may be a strict left-to-right or right-to-left row user, others prefer a diagonal strategy, and others may prefer to introduce designs in the tip boxes (initials or emoticons) with their tip use. Just go with whatever you find the most comforting or the technique with the highest rate of success on your reactions.

Prepare a master mix for multiple reactions.

The more times you have to pipet anything, the more potential error you introduce. Reactions become inconsistent across different samples. The way to avoid wearing out your opposable thumb doing tedious, pointless and downright erroneous pipetting is to prepare a master mix. This is just a scaled-up version of your reaction to accommodate as many samples as you have. Multiply the reagents in a single reaction by (x + 1), where x is the number of reactions you really need. Again, there is always pipetting error and even with the master mix, you will end up short on volume if you only use ‘just enough’. Once the master mix is prepared and gently mixed (very important in this scenario or some of your reactions to do not get enzyme), pipet out equal amounts of your master mix into individual reactions. In this case it is sort of OK to break rule #1. Usually the only difference among your many samples is the DNA or other reagent that’s not the enzyme. In this case, add the enzyme second-to-last, divide the master mix among individual reactions and then add the variable reagent. This variable reagent is usually such a small fraction of the total reaction volume that your enzyme is still under safe buffer conditions. Feel free to come up with your own ‘lucky number’ for scaling up your reactions with more than enough volume to accommodate the reactions you really need.

Avoid gel purification of DNA fragments if at all possible.

Sometimes you may need to isolate a DNA fragment for molecular cloning purposes. Agarose gel purification is a way of doing that. Companies will sell you easy-to-use kits to do this. In my experience, the sample losses are so great that it’s not even worth it. It is very difficult to obtain the quantities of DNA you need for subsequent steps from gel purification. At that point, you can either resort to faith-based cloning, in which, you can’t see your fragments on analytical gels with the human eye nor with help from the imaging camera, but you use it for ligation any way. Some times this works, but usually you are just disappointed when you check your transformation results. There are other tricks to avoiding this technique and if you are a clever cloner, you can get around having to gel purify anything. I swore it off several years ago and have never looked back. Sometimes, gel purification is unavoidable and I would recommend invoking some kind of Voodoo incantation to help success along.

I’m not convinced that the enzyme ligase does anything, but add it anyway.

Ligase is the enzyme responsible for gluing two desired DNA fragments together. The stitching together of these molecules in vitro is not the most efficient process, but we’ve been told that ligase ultimately seals the deal between our pieces of interest. Let’s just say, I’ve done enough positive and negative control reactions (more on that below) and I’m not sure ligase really does anything. However, I add it anyway because… protocols. Really, at this point in the cloning process, you’ve done so many purely superstitious acts, it doesn’t matter if you do one more. UPDATE: As per the comments below, Yes, ligase really does do something. I’m just convinced that it knows which pieces I want glued together and which ones I don’t. It always seems to favor the ones I don’t want, but just enough so that I still check through quite a few possible clones before I find the right one.

Use restriction enzymes until you have evidence they don’t work anymore.

These enzymes are the molecular scissors that cut DNA. They expensive, but they can last decades past their expiration dates when they are properly taken care of. Y’know, when you keep them cold, don’t vortex them or contaminate them with other reagents. You will also have to keep them in a frost-free freezer so they do not endure the temperature fluctuations of a self-defrosting freezer. So until you have evidence confirming a failed digest, keep using the enzyme.

Do the controls.

This rule is true across all scientific disciplines, but in the case of molecular biology work, it can save you lots of time, headaches and wasted reagents. For anything that you are doing, make sure you do a negative control that you know shouldn’t work and a positive control that should work. If both of these types of reactions give the expected result, then you know how to interpret all of your other samples. If only one or the other or neither of these controls work, then you will have difficulty saying what is going on with your experiment. It usually means there is a problem with user error or some other fatal flaw in your construction plan. You may try to interpret faulty experiments and even hope against hope that your experiment worked, but this relies more on faith and superstition than scientific probabilities.

Phosphatase treat your vector or insert but not both.

In many molecular cloning experiments, you are trying to combine two separate pieces of DNA with one another (a vector and an insert) in a useful way. However, sometimes there’s nothing or not much to stop the ligase enzyme from gluing together the vector with itself or the insert with itself, giving you useless byproducts. This is what the phosphatase enzyme does. It can remove the reactive chemical group from a piece of DNA such that ligase can’t use it. So if you treat one piece of DNA but not the other, it eliminates the possibility that ligase will glue any piece to itself, but instead glue the two pieces together. However, if you ligate both pieces, they all become useless to ligase. Sure, I didn’t think it was doing anything anyway, but now those reactions are guaranteed not to work.

Selection > Screen > Hope

When you are trying to get organisms to produce the DNA construct that you have engineered, it is better to select than to screen and better to screen than hope with blind faith. When you are selecting for a construct, all of the cells with the wrong thing will die and only cells with the right DNA will live. Thus, anything living at the end of that experiment is likely to be correct. When you can’t do this, there are ways of screening either based on color or replica plating onto a special medium. The most widely-used example is blue/white screening. If your bacterial colonies are white, they have the correct DNA. If they are blue, they do not. This color gives you a visual clue as to which colonies are most likely to give a positive result based on another experiment. If you can do neither of these things and can only hope to find the correct clone in a plate with hundreds of colonies to choose from, then you have more work ahead of you. In any event, doing the controls is still important. In the case where neither selection nor screening is possible, it may not even be worth looking through the colonies with subsequent experiments to verify a positive DNA sequence. There may just be too many false clones to sort through. Nevertheless, you may try it anyway. 99% of the time you will just end up wasting reagents and time. There does exist a possibility that the correct one can be found, and if you find it, you should probably buy a lottery ticket on the way home.

Sequence your finished construct to verify it is error free.

Once you think you are done with piecing together the DNA sequences you need, you will want to perform experiments to verify that the pieces have come together as you intended. Sure, you’ll cut them again with enzymes and run them on a gel to make sure it looks as expected, but you need to sequence the DNA to make sure that no point mutations have been introduced somewhere along the line. The enzymes responsible for copying the DNA pieces along the way have error-checking features to maintain sequence integrity, but the course of a typical molecular biology project will involve such a length as to make a sequencing error a formal possibility. To assume correctness is just hubris. Sequence it to make sure. Data always trumps assumptions, and that’s no superstition no matter what your scientific discipline.

Make freezer stocks.

Once you have your precious construct and are sure that the sequence is error free, you will want to get to work on your exciting new experiments so you can get groundbreaking results, publish a paper in a high impact journal, secure your own funding, get a job offer at a top research institution, win the Nobel Prize and ride off into the sunset on a unicorn.* But first, YOU MUST MAKE A FREEZER STOCK OF THE CELLS CONTAINING YOUR PRECIOUS DNA CONSTRUCT. Seriously, if you don’t, I’ll make you walk the plank. Make several tubes; label them with an identifiable name (pNobel2015), include the date and any antibiotic resistances. Then write all that shit down in your notebook. For extra credit, generate a graphic map which notes the important features of the sequence. Put the freezer stock tubes in your own freezer box as well as the lab repository. If you don’t do it now, there is a high probability you will forget to do it. You may have to kiss your Nobel prize goodbye if your peer reviewers ask for additional experiments that may require you to go back and use the DNA and your cells are too old to resurrect. Well, maybe not, but I guarantee there will be some poor graduate student or postdoc will carry on the torch of your research several years after you made the construct and it is nowhere to be found. Thanks to you slacker, they’ll have to remake the whole thing from scratch. You did write down your primer sequences, right?** Just save the world a lot of trouble and make the freezer stock.

I’m sure there are many more guidelines that other cloners could add. Maybe we should all just resort to Gibson cloning methods now anyway. Feel free to add your own guidelines in the comments section.

Johnna

*Just me? OK then.

**So help me God, if you didn’t… Chickens are being sacrificed to empower Voodoo dolls of you to exact vengeance.

Do you want to make a plastid?

FrozenNext up in the Frozen series… Do you want to make a plastid? Here’s the snowman version from Frozen for your reference.

Plant cells…

Do you want to make a plastid?

Come on, differentiate

Time to photosynthesize

Special things to metabolize

C’mon they need a fate

Those used to be proplastids

And now they’re not

We hope genetics tells us why!

Do you want to make a plastid?

It doesn’t have to be a chloroplast

Do you want to make a plastid?

Some grana stacks green and tall?

I think some development is overdue…

Environment gives you cues

From the nucleus transcription calls!

(Induce there GLKs)

It gets a bit confusing

All these proteins whizzing by

Translocation…

TIC, TOC, TIC, TOC, TIC, TOC

Chromoplasts get lots of color

With all these carotenoids

Amyloplasts store starches

For all the tissues underground!

Do you want to make a plastid?

It could even be a tannosome

Yes, etioplasts are in there

In the dark since who knows when

Shine the light to start,

The nucleus is there for them,

No longer inside meristems,

Time for some changes to begin!

They do so many functions,

Biochemistry

What can’t plastids do?

Do you want to make a plastid?


Well, if you are a plant cell, the answer to that refrain is always, “Yes!” Plastids are specialized organelles in plants that perform a wide array of specialized biochemistry. The most well-known and recognized of these are chloroplasts. These subcellular compartments are packed full of the thylakoid membranes and enzymes necessary to convert light into sugars. Chloroplasts perform other important biochemical reactions including the synthesis of starches and fatty acids as well as the assimilation of nitrogen into amino acids.

The chloroplast by User:Miguelsierra, adapted by User:Vossman via Wikipedia

There are other types of plastids made in plant cells for other specialized purposes. There are chromoplasts, which are full of carotenoid and other pigments giving them distinctive colors. The cells of tomato flesh, carrots and rose petals contain this type of plastid. Of course, pigments don’t do much good in root tissues. You may not find chloroplasts or chromoplasts there, but they do contain leucoplasts (loosely defined as non-green plastids used for storage of starches and oils). Storage-type plastids aren’t relegated to the roots. Recently other types of plastids have been identified, which specialize in the synthesis and storage of distinctive metabolites. Tannosomes have recently been characterized as specialists in tannin storage. A phenyloplast was also recently described in vanilla fruit as the storage location for vanillin precursor metabolites. In fact, the range of biochemical specialization that plastids perform strains their traditional nomenclature and definitions. As scientists learn more about them, the divisions between traditional types seems to blur. It seems that plants can make them as specialized as they need to be along a gradient of biochemical functions.

Types of plastids by Mariana Ruiz Villarreal LadyofHats via Wikipedia

Plant cells have definitely capitalized on the versatility of the plastid organelle structure to ensure dedicated locations for all manner of biochemical reactions and/or storage of their products. This is no small task. It requires the coordination of genes in two different genomes (that of the plant cell nucleus as well as a small genome in the plastid), trafficking of proteins throughout the cell, sensing of environmental cues, and synchronization with the overall plant developmental program. It’s enough to make any project manager run out of color-coded post-its and lose track of scheduled task times. Here are the highlights:

First of all, plastids do not exist in their mature forms in plant seeds and developing meristem tissues, but as immature and developmentally-flexible proplastids. These proplastids may not be special biochemists in their own right, but they have the potential to become any kind of plastid the plant cell needs. Consequently, proplastids have to know whether they are inside a root cell, petal cell or leaf cell. This means that plastid differentiation (aka development to maturity) must be coordinated with the development of the rest of the plant. Scientists are beginning to learn more about how this works, but this research is difficult since we’re talking about tiny, unremarkable pro-organelles within small portions of tissue. It’s a universal rule of cell biology that studying on small things is much more difficult than studying large things. Genetics has provided some new insights as to which genes have control over this developmental process. Transcription factors like the Golden-like (GLK) family of proteins have been identified as major players in this process, turning on suites of other genes to trigger proplastid development into mature plastids.

Of course, a lot of plant cell and plastid development is tied to environmental cues like light intensity. For example, plants can develop up to a point in darkness, but that doesn’t mean those cells have the same mature chloroplasts present in light-grown plants. Dark-grown plant cells contain plastids termed etioplasts, which are somewhat more differentiated than proplastids but not quite chloroplasts. Upon illumination, etioplasts quickly whip up thylakoid membranes and the photosynthetic machinery and get to the work of photosynthesis.

Plastid development and maintenance requires a large protein migration within plant cells. While plastid genomes make a number of their proteins, they do not make all of their resident proteins. Many essential proteins must come from genes transcribed in the plant cell nucleus and translated in the cytoplasm or endoplasmic reticulum. Thus, plant cells have an elaborate system of trafficking proteins to the proper cellular destination. This is true for all kinds of eukaryotic cells, but plant cells have extra organelles to distinguish among relative to animal cells. For those proteins with final destinations in the chloroplast, they have signals within their sequences that target them there. When they arrive at the chloroplast, they must pass through the surrounding double membranes using the Translocon of Outer Chloroplast Membrane (TOC) and the Translocon of Inner Chloroplast Membrane (TIC). In addition to the large influx of proteins that must be deposited upon initial development, the nucleus continues to provide essential proteins to the chloroplasts throughout their lifetime. It’s a constant flurry of activity maintained by two-way communication between the nucleus and chloroplast such that each organelle knows the biochemical status of the other.

Development into a mature plastid type isn’t the end of the line. There is still some developmental plasticity allowing certain plastids to convert into other plastid types. I’ve mentioned before that chloroplasts convert to chromoplasts in ripening fruit, as can be seen in the characteristic color change of green, unripe fruits into bright yellows, oranges and reds. Chloroplasts also provide the starting point for the development of other plastids specializing in storage of certain biomolecules. Plant cells also have ways of recycling the valuable contents of their plastids when cells and tissues reach the end of their lives (think autumn leaf color-change). As you might expect, this process requires more changes in gene transcription, protein synthesis and membrane structure orchestrated by the cell nucleus in response to more environmental and developmental cues.

Finally, proplastids and plastids of all types have ways of replicating themselves within plant cells. In this way, as plant cells divide each new daughter cell will contain roughly equal numbers of plastids. It wouldn’t be very good if a certain cell line within a tissue ran out of plastids. As much as plastids rely on the nucleus to full development and function, the nucleus cannot spontaneously create plastids from nothing. This wrinkle adds another layer of complexity to plastid biology. It means plastids must have some way of dividing themselves within the cells, and when plant cells divide, they must have some way of equally segregating the multiple plastids between the two new cells.

Basically, plastids are a lot of work for plant cells to make and maintain. However, these high-maintenance organelles are worth it given the biochemical rewards they offer. Scientists are only beginning to understand how plants make, maintain and recycle their plastids. Pieces of the complex system are studied by plant biologists all over the world and will likely keep them busy for many years to come.

 

Johnna

 

References and Links:

http://www.disneyclips.com/lyrics/frozenlyrics2.html

http://en.wikipedia.org/wiki/Plastid

http://link.springer.com/book/10.1007%2F978-3-540-75376-6

http://www.springer.com/life+sciences/plant+sciences/book/978-94-007-5723-3

http://www.uoguelph.ca/~itetlow/publications/assets/pdf/BY008-03-60-125.pdf

http://pcp.oxfordjournals.org/content/51/10/1601.long

http://www.ncbi.nlm.nih.gov/pubmed/23851381

http://www.ncbi.nlm.nih.gov/pubmed/23876238

http://journal.frontiersin.org/Journal/10.3389/fpls.2014.00124/abstract

http://onlinelibrary.wiley.com/doi/10.1046/j.1365-313X.2002.01390.x/full

http://aobblog.com/2013/09/announcing-brand-new-cellular-organelle-tannosome/

http://www.ncbi.nlm.nih.gov/pubmed/24683183

Prokaryotic Picassos

Photosynthetic organisms are brilliant artists when it comes to their use of colors. Most people are familiar with the wide range of colors plants display. Roses are red, violets are blue, anyone? Well, cyanobacteria package prodigious amounts of pigments in prokaryotic form. Their name is derived from their blue-green color. It’s kinda their trademark. As photosynthetic organisms, they have plenty of chlorophyll in their photosynthetic machinery. Their blue color comes from their light-harvesting antenna, the phycobilisomes (PBS), which are comprised of proteins with blue pigments.

Phycocyanoobilin from Wikipedia

Phycocyanin from Wikipedia

Phycobiliproteins bind blue bilin pigments and assemble into discs; these discs assemble into the large phycobilisome complexes that sit on top of the photosystems and funnel light energy to them. The PBS looks like a giant blue octopus with a central core and a number of ‘arms’ of phycobiliprotein discs that extend out in all directions. Because these complexes are particularly good at absorbing orange light, which chlorophyll does not, the PBSs extend the wavelengths of light (and therefore the energy) available to cyanobacterial photosystems.

 

Phycobilisome General Structure

Some cyanobacteria like Fremyella diplosiphon can take color to another level. They can change the composition of their PBSs according to the color of light available to them in a process called complementary chromatic adaptation (CCA). This is possible because some species of cyanobacteria have two varieties of phycobiliproteins, each of which coordinates a distinct pigment. Phycocyanin binds the blue phycocyanobilin chromophore, which efficiently absorbs orange and red light and appears blue in color. Phycoerythrin binds the pigment phycoerythrobilin, which efficiently absorbs green light and appears pink to red in color. Thus, these cyanobacteria have blue PBSs in red light and have a typical blue-green appearance. However, in green light, they produce PBSs containing phycoerythrin and have a rose or red-colored appearance.

Phycobiliproteins, bilin variation, and group III CA regulation. (A) Phycocyanin and phycoerythrin (blue and red lines, and in vials) absorb in regions of the visible spectrum not well absorbed by chlorophyll or carotenoids. Attached bilins: PEB, phycoerythrobilin; PCB, phycocyanobilin. (B) Natural diversity in coloration of many different cyanobacterial species due to variation in their bilin content [photograph by Christophe Six. Reproduced with permission from Six et al. (2007) (Copyright 2010, Biomed Central Ltd.)]. (C) Group III CA regulation model for F. diplosiphon in red light, showing the asymmetric regulation of red-light active genes (orange) and green-light active genes (yellow) by the Rca and Cgi systems. Dashed line represents proposed repression by the Cgi system; yellow balls, phosphoryl groups; blue boxes, RcaC binding sites.

Phycobiliproteins, bilin variation, and group III CA regulation. (A) Phycocyanin and phycoerythrin (blue and red lines, and in vials) absorb in regions of the visible spectrum not well absorbed by chlorophyll or carotenoids. Attached bilins: PEB, phycoerythrobilin; PCB, phycocyanobilin. (B) Natural diversity in coloration of many different cyanobacterial species due to variation in their bilin content [photograph by Christophe Six. Reproduced with permission from Six et al. (2007) (Copyright 2010, Biomed Central Ltd.)]. (C) Group III CA regulation model for F. diplosiphon in red light, showing the asymmetric regulation of red-light active genes (orange) and green-light active genes (yellow) by the Rca and Cgi systems. Dashed line represents proposed repression by the Cgi system; yellow balls, phosphoryl groups; blue boxes, RcaC binding sites.

To achieve this dramatic color change, the cells have elaborate systems for sensing the incident light wavelengths in their environment and eliciting the appropriate response. Changing out the PBS antenna requires changes in gene expression of the protein components as well as the pathways that synthesize the necessary pigment (phycocyanin vs. phycoerythrin). It also means that the existing phycobiliproteins and their pigments must be degraded and their components recycled. The PBS antenna complexes comprise a significant portion of soluble protein within cyanobacterial cells, so it is important that the pieces be reused, no matter the light condition. All of these requirements add up to a sophisticated system of multiple players working to make this simple-looking color change happen on cue. This phenomenon is the research area of a number of groups working on cyanobacteria and photosynthesis.

greenlightPBS RedlightPBS

In this way, cyanobacteria are similar to Picasso, going through a blue period and later a rose period. In the case of Fremyella, the change is not due to swings in mood from melancholy to cheer, nor is it purely aesthetic. In biology, even beauty is functional. In the case of complementary chromatic adaptation, it allows cyanobacteria to most efficiently harvest the wavelengths of light available to them. You may be wondering when cyanobacterial cells would be exposed to differently colored light sources outside of an elementary school science fair project. Well, this scenario is more commonplace than you would think. For example, cyanobacteria cells close to the surface of a pond or ocean would be exposed to higher amount of red light compared to those deeper in the water column.* Those cyanobacterial cells that find themselves deeper in the water column would have much less red light because it does not penetrate as well as green light and other photosynthetic organisms living closer to the surface have already absorbed all of the red wavelengths of light allowing only green to pass through.**

Light microscopy image of a normal 'Fremyella diplosiphon' colony (green, left side) adjacent to a 'F. diplosiphon' mutant capable of only producing the light-harvesting pigment phycoerythrin (red, right side), growing on an agar plate in red light.

Light microscopy image of a normal ‘Fremyella diplosiphon’ colony (green, left side) adjacent to a ‘F. diplosiphon’ mutant capable of only producing the light-harvesting pigment phycoerythrin (red, right side), growing on an agar plate in red light. Credit: David Kehoe, used with permission

The image on the left may appear to be some kind of impressionism masterpiece or abstract work by a yet-to-be-discovered artist, but really it’s the combination of two cultures of Fremyella. The green side is the normal strain, while the red side is a mutant that contains only the red phycoerythrin pigment.

This post was written for the April Berry Go Round blog Carnival. Plants aren’t the only photosynthetic organisms that use color beautifully and efficiently.

 

 

 

Johnna

*For those of you paying attention, this means they would use the blue phycocyanin-pigmented PBSs and appear blue-green.

**This means that cyanobacterial cells deeper in the water would adapt to use phycoerythrin in their PBSs and appear red.

References and Links:

http://www.prl.msu.edu/faculty/montgomery_beronda

http://en.wikipedia.org/wiki/Cyanobacteria

http://www.pnas.org/content/107/20/9029.full

http://www.psi.cz/ftp/publications/others/david/Grossman2003.pdf

http://www.bio.indiana.edu/faculty/directory/profile.php?person=dkehoe

http://sites.bio.indiana.edu/~kehoelab/research.html

http://www.pnas.org/content/107/20/9029.full

http://en.wikipedia.org/wiki/Phycocyanobilin

http://en.wikipedia.org/wiki/Phycocyanin

http://en.wikipedia.org/wiki/Phycoerythrin

 

Heterotroph Denial

Alright readers, I have a lot to do today, but I feel compelled, nay, obligated to blog about something buzzing around the interwebz lately. Remarkably, no- this isn’t about plants or algae or any other autotroph. Confused since this is an all-autotroph blog? After reading a news article that Ukrainian model Valeria Lukyanova believes she can subsist on only air and sunlight, I have to set the record straight. Let me say this slowly so there is no confusion and please feel free to share this with your friends.

You. Are. Not. Photosynthetic.* Humans are heterotrophs. Full Stop.

It’s really important to me that you know this.

I hope that some of the links on my Basics page will explain why this ‘Breatharian’ lifestyle is crazy. Yes, sunlight is the main source of energy feeding into the Earth’s biosphere. This makes the sun both physically important to our survival and an intimate part of our human culture. The sun is critically important to the way we live because our lives depend on photosynthetic organisms. This is because truly photosynthetic organisms have biochemical machinery that can capture and convert the sun’s energy into useful energy storage molecules (ATP, NADPH, glucose). Even photosynthetic organisms require water for this process. Why do you think people get so up in arms about droughts?

Let’s consider the implications of a Breatharian reality.  If Breatharianism were possible, then why are there more than 800 million food-insecure people around the globe? Surely, they have access to sunlight. Should we just tell them to quit complaining and enjoy their sun? If this were the case, then agriculture is the biggest con of all time. So, let’s just not do it anymore. The food service industry is just profiteering off of the heterotrophic hoax as well. This conspiracy was all masterminded by some elite group of gardeners**, hunters and herders that swindled early humans into buying otherwise worthless crops, meat and dairy. Once the human population was hooked on the delicious calories, those robber barons have been laughing all the way to the bank. I’m quite sure that must’ve been how it all went down.

A brief on-line search reveals that Valeria is not the only adherent to this practice. Sigh. Sure, the human body is amazing and can endure periods of time without food and water, but our bodies cannot survive indefinitely without them. Check out the video below for a breakdown of what happens on a Breatharian lifestyle (aka dehydration and starvation).

I know I’ve mentioned KungFu Panda before, but this scene is worth mentioning today:

Tigress: It is said that the Dragon Warrior can survive for months on nothing but the dew of a single ginkgo leaf and the energy of the universe.

Po: I guess my body doesn’t know it’s the Dragon Warrior yet. I’m gonna need a lot more than dew and universe juice.

Yeah, Po. Me too.

Fasting is a common religious practice, but believing that one could solely subsist on sunlight and air as a lifestyle choice is just downright heterotroph denial. This post topic gets a special new category for blog posts here (head-desk). Respect what real photosynthetic organisms are doing for you today… and eat one of them. Then wash it down with the aqueous solution of your choice. I really hope we don’t have to cover this again.

Johnna

* Not even those sea slugs turned out to be photosynthetic. They looked like leaves crawling around the ocean floor. Srsly? What makes you think you will fare any better. Heterotroph = you have to eat something else.

** Yes, I seem to remember something about this in the Master Gardner handbook. Oops, I guess I let the cat out of the bag. Guess I won’t be getting my new card this year.

References and Links:

http://www.huffingtonpost.com/2014/02/28/breatharian-barbie-valeria-lukyanova_n_4873706.html

http://www.theguardian.com/world/2013/jun/14/living-on-light-without-food?guni=Article:in%20body%20link

http://science.howstuffworks.com/innovation/edible-innovations/breatharian.htm

http://en.wikipedia.org/wiki/Inedia

http://www.thedailybeast.com/articles/2014/03/02/introducing-breatharianism-the-dumbest-diet-of-all-time.html

Hot Peppers under the Microscope

Something new under the sun… pepper pigment packaging.*

pepper

Just when you thought you had seen the last of red hot chile peppers in the Superbowl halftime show yesterday**, I’m still talking about Capsicum science. The other pepper-themed posts have been all about the heat, but capsaicin isn’t the only chemical these plants make. Capsicum species are also great at producing nutritious carotenoids. Again, because Capsicum species grow well in arid environments, they offer an advantageous platform for providing these nutrients in an efficient way. In order to get our peppers to pack more punch in the nutrition department, we must better understand the metabolic machinery that makes these colorful molecules.

Carotenoids are the pigments that give us the yellow, orange and red colors in our peppers. You may be familiar with the common beta carotene molecule that is the precursor for vitamin A. That’s just one pigment. Plants and peppers, in particular, are adept at making a wide range of colorful carotenoids. However, all peppers start out green because the cells of the fruit contain photosynthetically active chloroplasts. As the fruit matures, these green chloroplasts undergo a major developmental change to become colorful chromoplasts. This process involves changes in gene expression, protein function, membrane structure and overall metabolism. By the end of the transition, chromoplasts are filled with an array of carotenoids, giving the fruit its hallmark red color.

Today’s journal club features a recent paper by Kilcrease et al that explores pigment localization within the chromoplasts of living plant tissue. This combines the Capsicum expertise of New Mexico State and the hyperspectral imaging capabilities of the Timlin lab at Sandia National Labs.*** It turns out that the different pepper varieties make characteristic carotenoids and these are made/stored in specific intracellular sites.

Here’s how the science breaks down:

Observations: For this example, observations are coming from two different directions (pepper pigment biology and spectral imaging method development).

Different pepper varieties produce different arrays of carotenoid pigments in their mature fruit. The literature suggested that there were significant differences in chromoplast morphology among these varieties. Some experimental evidence suggested that certain pigments were so concentrated they formed crystals within the plant cells.

Hyperspectral confocal Raman microscopy**** can provide chemical information in high resolution on living plant tissue. The molecular structure of chemicals like carotenoids makes them straightforward to identify. Using multivariate image analysis, a spatial model can be generated to show where the chemicals are within the microscopic image.

Hypothesis: Hyperspectral confocal Raman microscopy can be used to determine the carotenoid localization within the ripe fruit of different pepper varieties. This data will show whether or not the pigments are localized in distinct places within chromoplasts.

Experiment: Researchers analyzed the tissue of 5 different hot pepper varieties (mature fruit; and no, they didn’t use Bhut Jolokias or Scorpion peppers) using four different microscopic techniques (scanning electron microscopy, transmission electron microscopy, laser scanning microscopy, and hyperspectral confocal Raman spectroscopy) to identify subcellular localization of carotenoid pigments. The pigment content of each of the peppers was also analyzed using the analytical chemistry technique HPLC.

Results: The different types of peppers analyzed in this study varied considerably in carotenoid composition, chromoplast structure and sublocalization of carotenoids. The pigments did localize to specific sites within the chromoplast as well as some subcellular lipid bodies outside of the chromoplast.

Conclusions: The combination of these methods allowed for the more complete characterization of chromoplast structure and pigment composition in five different pepper varieties. These data can serve as new traits when considering breeding peppers for increased nutrient content.

Think Ahead:  The example the authors give is for aiding in the breeding of superior chiles in terms of increased carotenoid content. For example, a variety with high carotenoid content can be crossed with one with large chromoplasts to potentially yield offspring with even more carotenoids filling the larger chromoplasts. In this way, the results from these analyses will provide new molecular traits characteristic of certain pepper varieties. Genetics can then be used to mix and match those traits in desirable ways. Also, all of these experiments were performed on fruit that was already at a certain stage of ripeness. It will be interesting to perform an extended analysis on fruit as it ripens from green to red. This kind of time course experiment will yield more information about how these specialized synthesis and storage sites for the different carotenoids form as the chromoplast develops.

Ultimately, knowing more about how peppers make their carotenoids will allow scientists and breeders to develop more nutritious plants. This means not only understanding the chemical synthesis of these molecules, but also how the plant cells physically/spatially accommodate the increase in those metabolic pathways.

Johnna

*Say that fast 3x!

**It was a weird pairing of performers, but ‘Give it away’ still rocks IMHO.

***The second author on this paper is Aaron Collins (Sandia National Labs), an old grad school and photosynthesizer colleague of mine from WashU. He mentioned this project to me at a meeting last summer and bragged about the benefits of collaborating with biologists working on Capsicum (=pepper perks!).

****Yes, as fancy as it sounds.

References and Links:

(Caution: paywall for full text) http://www.ncbi.nlm.nih.gov/pubmed/24118159

Here’s a nice research highlight with the Raman microscopy figure) http://parc.wustl.edu/files/parc/Timlin_PARC_EFRC_Research_Highlight_September_2013.pdf

For more on hyperspectral imaging: http://www.sandia.gov/bioenergy-biodefense/timlin.html

For more on chile peppers: http://www.chilepepperinstitute.org/

Mass out of Thin Air

“True, I talk of dreams,

Which are the children of an idle brain,

Begot of nothing but vain fantasy,

Which is as thin of substance as the air…”

Romeo and Juliet, William Shakespeare

It’s true the atmosphere around us is invisible, but is it really as insubstantial as Mercutio thinks? For humans perhaps, but not for photosynthetic organisms. Sure, plants may appear to be firmly rooted in the ground, but this tether to substance is only a deception. They really make a living knitting mass out of thin air by pulling carbon dioxide from the atmosphere and fixing it into useful biological molecules.*

General Sherman Tree via Wikipedia

This feat is most obvious in the world’s most massive organisms- the Sequoias. These giants are experts at silently converting air into substance. Tree size can be measured in different ways like height or girth, but the true measure of mass is trunk volume. Based on this measure, the largest tree ever reported was the Lindsey Creek tree. It was felled by a storm in 1905, and the local paper published some details on its measurements to give an estimated trunk volume of 90,000 cubic feet. The Crannell Giant was discovered in 1926 and more precise measurements calculated a minimum trunk volume of 61,573 cubic feet. It was logged in the late 1940s.

The most massive living tree (single stem) is the giant sequoia (Sequoiadendron giganteum) General Sherman in the Giant Forest of Sequoia National Park with an estimated trunk volume of 52,508 cubic feet. It soars to a respectable 274.9 feet in height with a base circumference of 102.6 feet and is estimated to be more than 2,000 years old. It is truly the biological manifestation of majesty.

Of course, even this mighty giant has humble beginnings. Check out this video that documents the growth of sequoia seedlings over the first five years of their lives. You may still doubt how those young trees could grow into a giant the size of the General Sherman, but over the course of ~2300 years the area corresponding to the base of the General Sherman received 341 Gigawatt hours-worth of incident solar energy.** What would you do with that much energy? Well, you could power New York City for about a month. If you are a sequoia, you could use photosynthesis to turn it into ~50,000 cubic feet of tree.

Don’t think that an older tree like the General Sherman is going to sit on its laurels when it comes to gaining mass. As I mentioned yesterday, new research has shown that this kind of older tree is increasing its growth rate when it comes to adding mass. The statement can seem quite intangible when juxtaposing the most massive living organism and a substance as thin as air. How much mass are we talking about? The 40 cubic feet of growth General Sherman adds annually at 13.42 pounds of carbon per cubic foot corresponds to 537 pounds of carbon. That’s just over a quarter ton of carbon extracted from the air each year by a single tree.

With those numbers, I’ll have to disagree with Shakespeare. It seems that air has more substance than Mercutio would lead you to believe. Perhaps there is more to that dream as well.

Johnna

* Remember the equation for photosynthesis? No? You must be new here.

**∏(5.5 m)2 = 123 m2 x 4.2 kWh/day x 365 days/yr x 2300 yrs = 341GWh

The incident solar radiation on the area of the size of the base of the General Sherman. Sure, the size of the base would be changing over the course of those 2300 years, but the canopy area is much larger and the error bars on the age are on the order of centuries, so I’m calling it close. If anyone else would like to come up with a better algorithm that takes into account the changing area over the course of the tree’s lifetime, please show your work in the comments section below.

References and Links:

http://en.wikipedia.org/wiki/Sequoiadendron_giganteum

http://en.wikipedia.org/wiki/Lindsey_Creek_tree

 http://www.mdvaden.com/redwood_crannell_creek_giant.shtml

http://www.landmarktrees.net/elam.html

http://en.wikipedia.org/wiki/General_Sherman_%28tree%29

http://www.nps.gov/seki/naturescience/sherman.htm

http://www.monumentaltrees.com/en/trees/giantsequoia/biggest_tree_in_the_world/

http://library.thinkquest.org/J002415/Famous_Trees/General_Sherman/general_sherman.html

http://www.sampsongroup.com/Papers/Monitoring%20and%20Measuring%20Wood%20Carbon.pdf