Friday, January 29, 2016

Species Spotlight: Soybean

Until I started studying soybean for my PhD, I only ever associated them with the fields lining the highways of the Midwest and delicious edamame.

But soybean is important for a lot more than just food. Today let's take a closer look at the species I'm currently studying.

Soybean's scientific name is Glycine max. It originates from China and has been a successful agricultural crop in many countries. In the United States in 2012, soybean was the second most valuable crop right behind corn! There are dozens and dozens of cultivars and these are sorted into maturity groups. Maturity groups are based on the changes in day length required to produce flowering in soybean and thus are related to the latitude in which the soybeans are best suited to grow.

Beyond its uses for food, soybean is incredibly important in many other industries, including biofuel, crayons, paint, wax, clothing, elevator oil, the list goes on. In fact the average soybean acre produces 44 bushels which can be used to create 8gal oil, 2500gal soymilk, 320,000oz tofu (640,000g protein), 66gal biodiesel or 82,368 crayons!
Indiana State Fair homage to soybean. Personal photograph
Another reason soybean is a popular crop is nodulation. Nodulation is the process by which specific bacteria colonizes the roots. During this colonization process, a nodule (they look like bubble protrusions) is grown from the roots which becomes bacteria new home. These bacteria fix nitrogen that is biologically unusable into biologically useable versions of nitrogen. This available nitrogen is used by the soybean and also replenishes the soil. Because soybean replaces nitrogen into the ground, it is very useful for renovating a field after a nitrogen consumer crop, such as corn. This is why when you drive down the road in the Midwest you often see a field of corn one season and a field of soybean the next.


Another type of soybean I work with is Glycine soja, which is thought to be the closest non-domesticated ancestor of the current soybean we all know and love. The importance of non-domesticated ancestors in research cannot be understated. Domestication drastically changes the species genotype and phenotype, often limiting them to very narrow results. As you can see in the image below, soybean has been bred to produce thicker stems and more vegetative tissue which leads to greater yield. While all of these are good traits, during this process other traits may have been lost. For example, non-domesticated soybean (G. soja) has been shown to be more tolerant to dehydration stress than domesticated soybean (G. max Chen et al, 2006). That is why we are studying both domesticated (G. max) and non-domesticated (G. soja) soybean side by side. Maybe we can isolate a trait to potentially breed back into domesticated soybean!

Glycine max (left) and Glycine soja (right). Personal photograph.


There we have a brief rundown on the value and awesomness of the soybean! For more information about the agricultural side of soybean, I highly recommend this book: Coolbean the Soybean. It is full of facts and geared for a grade school-middle school level. Their webpage also has some fun activities for kids.

References:
http://genezoo.missouri.edu/soybean.cfm
http://corn.agronomy.wisc.edu/Crops/Soybean/L004.aspx
http://ncsoy.org/media-resources/uses-of-soybeans/
http://agron-www.agron.iastate.edu/Courses/agron212/Readings/Soy_history.htm
http://www.agcensus.usda.gov/Publications/2012/
Chen Y, Chen P, de los Reyes BG (2006) Differential response of the cultivated and wild species of soybean to dehydration stress. Crop Science 46 (5):2041-2046 doi:10.2135/cropsci2005.12.0466 

Friday, January 22, 2016

How It Works: Chlorophyll Fluorescence the Basics

Photosynthesis is my favorite metabolic pathway. I love everything about it. Today's post is going to look at one really neat, non-invasive way scientists can monitor photosynthesis: chlorophyll fluorescence.

During photosynthesis, light excites electrons in the chlorophylls found in antenna and photosystem cores. These excited electrons are passed along and can be used in photosynthesis, however, very rarely is 100% of this energy used in this manner. Imagine for a moment that you are photosystem II and your mouth is the special chlorophyll that can pass the electrons along. Now, you have a few friends who hang out with you, they get to be the antennas (light harvesting complexes). Instead of electrons, your friends are going to pass popcorn at you. Only the popcorn that you catch in your mouth, gets to be used in photosynthesis. If your friends slowly toss one piece of popcorn at a time, you can probably catch 100% of the popcorn. But what happens when they all throw a piece at you? Or when they each start throwing handfuls? Think you could catch 100% of the popcorn? Probably not. Neither can the photosystems, which means this energy has to be released other ways.

man I really want to make a video out of this example now... but I digress.. back to business

There are 3 fates this energy can undergo: used in photosynthesis (photochemical quenching), emitted as heat (non-photochemical quenching), or released as a light particle (fluorescence). The fluorescence part can be measured and then used to calculate photochemical and non-photochemical quenching. This is done with a fluorometer. There are 2 main types of fluorometer's utilized in the study of photosynthesis: Fast Repetition Rate (FRR) and Pulse Amplitude Modulated (PAM). The difference is in the fashion the light is emitted but the data one can gather is similar. I spent my master's degree using PAM so I'll be using it as my reference.


Examples of PAM fluorometers. Walz Diving and bench top models. Personal images
The main function of the fluorometer is to provide light and measure the amount of fluorescence. Some quick and important photobiological terms:
  • Open reaction centers - Photosystems are ready to accept electrons
  • Closed reaction centers - Photosystems have accepted electrons and cannot take any more at the moment 
The state of the reaction centers are important. If they are all open, not a lot of energy is going to be reflected back (fluorescence). Conversely, if they are all or mostly closed, the amount of fluorescence will go up. With that in mind, let's look at mock up chlorophyll fluorescence trace and link each part to the corresponding photobiology.
Diagram of a PAM fluorometry trace. Orange stars indicate when light pulse occurs. Personal illustration.
Usually the sample plants have been dark adapted for at least 10 minutes prior to reading (if you look at the diving PAM image above you'll note the gray circles attached to the coral, those were our homemade dark adapters). This dark adaption allows all of the electrons in PSII to be passed through to the end of the electron chain, rendering all of the reaction centers open. The amount of fluorescence detected in the dark is the background level when all reaction centers are open and is designated F0 or minimal fluorescence.

The saturating pulse is then fired (orange star). This saturating pulse is a very strong light, providing a plethora of photons and rendering all of the reaction centers closed. The top of the peak, when all of the reaction centers are closed, is labeled Fm or maximal fluorescence.

At this point what is known as the actinic light is activated. The actinic light is a non-saturating, steady beam of light that allows photosynthesis to occur but does not saturate (close) all of the reaction centers. Each saturating pulse now results in a small peak that corresponds to photochemical quenching or the amount used in photosynthesis. The difference from the top of photochemical quenching peak (Fm')  to the original saturation pulse (Fm) is accounted for by non-photochemical quenching.

Non-photochemical quenching (NPQ) can be separated out into 3 different types. These can also be measured by fluorescence. The fastest, occurring in seconds to minutes, is called qE which involves the xanthophyll cycle which I've reviewed before. The second type, taking minutes to hours, called qT occurs when the light harvesting complexes attached to photosystem II move to photosystem I in an attempt to balance light capture and electron flow through the two systems. The slowest takes hours to days and is known as qI, or photoinhibition. Photoinhibition occurs when the reaction centers start becoming damaged and must be broken down to be repaired. hopefully I'll blog more detailed looks at qT and qI and if I do I'll update this with links.

From the image above it is impossible to sort out the 3 types of NPQ. However, by dropping the sample back into darkness with precisely timed saturating pulses, one can calculate the fraction of NPQ coming from each of the 3 types. Observe the diagram below. Notice that as time advances the peaks get higher and higher.  The differences in the height accounts for the fractions of each type of NPQ.
Diagram of a PAM fluorometry trace with NPQ parameters. Personal illustration

The final measurement I want to highlight from a chlorophyll trace is that of the photochemical efficiency of photosystem II, Fv/Fm. This is one of the most, if not the most, common fluorescence measurement I run across in the literature. Basically, this is a measure of the health of the photosystems. For example, an Fv/Fm of 0.986 would suggest that the photosytems are running at 98.6% efficiency. A high efficiency means everything inside photosystem II is working properly and precisely. In contrast, when Fv/Fm is low, say 0.687 or 68.7% efficient, the photosystems are most likely stressed and/or damaged. Fv/Fm is calculated by taking the variable fluorescence (Fv) and dividing it by the maximal fluorescence (Fm). The variable fluroescence is simply Fm - F0. As a note, if you ever come across it in the literature as Fv'/Fm' that simply means it was calculated from samples in the light. In the field, it is not always possible to allow a 10 minute dark acclimation period and the ' equals collected without dark adaption.

And that's the basics of how chlorophyll fluorescence works! There are a host of other parameters that can be calculated but the ones above are the ones I encounter the most in the literature. To sum up:
  • qP = amount of energy used in photosythesis
  • NPQ = amount of energy sent into qE + qT + qI 
  • Fv/Fm= efficiency ("health") of photosystem II
Chlorophyll fluorescence is a powerful tool for photobiological research. There are many different models that can be used in the lab or in the field, the prices range from not bad for lab equipment to wow ouch, and the best part it does not harm the plant in anyway.

References
Falkowski and Raven, 2007. Aquatic Photosynthesis: Princeton University Press
Goss and Lepetit, 2015 Biodiversity of NPQ. Journal of Plant Physiology 172:13-32

Friday, January 15, 2016

Best Biochemist: Xanthophylls & the Xanthophyll Cycle

My last post was a brief overview of photosynthesis. Today, zoom into photosystem II to examine how accessory pigments help with light capture. Photosystem II is typically shown in the textbooks as an oval, with the oxygen evolving complex occasionally added as an external oval. The problem with this is that it pays zero attention to the large antenna system that exists with the depicted photosystem II core.
Looking down into a membrane a rough sketch of PSII core (green) surrounded by the various antenna complexes.
Personal illustration

These antennas are several other protein complexes called Light Harvesting Complexes (LHC). The LHC have many chlorophyll and accessory pigments that can absorb light. The energy absorbed is then to their neighbor chlorophylls all the way to the special chlorophyll in the reaction core so that ATP can be made. Think of these pathways as a line of people standing next to each other, shoulder to shoulder, one standing at the goal line. Balls (photons) are tossed randomly at the line and whoever catches one can pass it down along the line of people to the goal. The greater amount people in the line, or even lines, the higher percentage of balls they will catch. For the plant, the increase in surface area due to the LHC allows a high percentage of light to actually reach the goal line of photosystem II.

Carotenes and xanthophylls are two important light capturing pigments. They extend the wavelength range of light the plant can use. Previously, we looked at carotenes. There is only difference between carotenes and xanthophylls.  Carotenes are made of only carbon and hydrogen, while xanthophylls also contain oxygen. All xanthophylls are derived from carotene precursors in the same fashion as described in my previous post. A few specific xanthophylls are incredibly important for photosynthetic regulation. Their names are: violaxanthin. antheraxanthin, and zeaxanthin. But as my son would say, we'll just call them V A Z "for short".  

Photosynthetic stressors generate excess amounts of reactive oxygen species, including singlet chlorophyll. This decrease the pH triggering  the conversion of V to Z. The conversion happens quickly, allowing the plant to get rid of excess energy before it can cause irreparable harm. 

Violaxanthin cycle Yikrazuul Public Domain Wikipedia
Notice in the above diagram that  only difference between these is the presence of oxygen (O) or a double bond on the terminal rings. This is a minor, but incredibly important, difference. Z, with its lack of oxygen, can accept electrons from singlet chlorophyll radical. A singlet chlorophyll has been excited by light, but for some reason that excited electron is not able to be passed to the photosystems. This excited electron must be sent somewhere, and that's where Z comes in. The xanthophyll cycle plays a major role in non-photochemical quenching, the term for energy absorbed but not used in photosynthesis.

Another type of non-photochemical quenching involves LHCII aggregation. Aggregating LHC results in the removal of antenna from PSII reducing damage to the core. This occurs when more Z than V is present in the membrane. The mechanism behind this was examined in a paper that came out this month (Janik et al 2016). Both V and Z can bind to a pocket in LHCII which causes either the stabilization or destabilzation of LHCII complexes. When light is high, stress is high, and more Z is made resulting in more Z in the LHCII pocket which destabilizes the LHCII trimers into monomers. The researchers suggest that this could allow the monomers to form an as of yet unknown complex of LHCII that increase quenching. It is an interesting proposition.

Regardless of how it works, xanthophylls are critical for the survival of plants due to their ability to remove reactive oxygen species formed during normal, but especially stressed, photosynthesis.

References

Friday, January 8, 2016

Photosynthesis Overview

One of the defining characteristics of plants is their ability to produce their own food via photosynthesis. Photosynthesis is also responsible for all of the food we eat! Almost all of the biological carbon on this planet is generated by photosynthesis. This carbon is created from carbon dioxide, which is a waste product we breath out. And the oxygen that we breath in, we also have photosynthesis to thank for that. After all, oxygen is a waste product of photosynthesis. But how does this all happen? And where does it happen inside the plant? Let's find out!

When thinking about photosynthesis, many people associate it with the green color of plants. This green color is from chlorophyll, a pigment utilized in one small section of photosynthesis. Photosynthesis occurs within the chloroplast organelle inside plant cells. Within chloroplast are stacks of thylakoid membranes (in green below), which house all the photosynthetic machinery. In fact, chloroplasts have their own genome and most of the important photosynthetic proteins are created in-house, so to speak.


Chloroplast By SuperManu • CC BY-SA 3.0 • Wikipedia
Photosynthesis is typically broken down into two phases: light-dependent and light-independent. You may have heard of these as the light and dark reactions of photosynthesis. The trouble with this nomenclature is that the dark reactions occur only IN the light, they just do not USE light directly. Thus the naming change to light-dependent and light-independent. Each of these reactions take place either on, or inside, the thylakoid membranes.

Photosynthesis by The Awkward Yeti
The overall reaction of  photosynthesis: light + carbon dioxide + water -> glucose + oxygen, exactly as depicted in the awesome The Awkward Yeti cartoon above.
  • Light-dependent = light + water -> oxygen + ATP (cellular energy) + NADPH (electron carrier). 
  • Light-independent = ATP + NADPH + carbon dioxide -> glucose
Thus both light-dependent and light-independent reactions play important and linked roles in photosynthesis. Let's take a quick look at both "sides" individually.

Light-dependent


Light-dependent reactions occur on the thylakoid membrane. Being on the membrane is critical because the key to ATP creation is the establishment of a proton gradient. Just like water will only flow downhill, molecules can "flow down" a membrane from areas of higher to lower concentration. In this case, pumping protons to side A results in a lower concentration on side B. These protons then be passed through from side A to side B via a special protein that spans the membrane called ATP synthase. ATP synthase, just like its name sounds, generates ATP. But how are these protons continually passed to side A (lumen) so that side B (stroma) always has less? This is accomplished by the photosystems.
Thylakoid membrane 3 by Somepics • CC BY-SA 4.0 • Wikipedia
Photosystem I (PSI) and photosystem II (PSII) both capture light at slightly different wavelengths with special chlorophyll molecules. An electron inside these special and specific chlorophylls is excited by the light and "jumps" out of the chorophyll into Q. Q carries the electron out of PSII and into the plastoquinone pool. This leaves the chorophyll without an electron, a very dire situation! This electron is replaced by breaking up water and taking an electron from it. After 4 of these cycles, a single molecule of oxygen and 4 protons are generated.

The excited electron takes a ride through a few electron carriers, including cytochrome b6f. Cytochrome b6f releases the proton that was riding along with the electron into the lumen, while passing the electron to plastocyanin. Plastocyanin will ferry the electron to photosystem I.

As stated earlier, PSI has special chlorophyll molecules that can be excited by light. This excited electron jumps up in energy and is passed along to create NADPH. But unlike PSII, photosystem I does not have an oxygen evolving complex to replace this jumping electron. It is replaced by the electron carried in by plastocyanin. The replacement electron originated in photosystem II.

The movement of electrons from photosystem II -> photosystem I -> NADPH is called the Z scheme.

Z scheme - personal illustration

It is traditionally drawn as I did above, which looks more like an N! But when it was originally described, prior to the addition of energy as the X axis, it was drawn as a Z from PSII --> PSI.

Light-independent


Also known as the Calvin cycle, after Melvin Calvin whose work in uncovering this pathway earned him the Nobel Prize in Chemistry. The Calvin cycle is a great big circle during which carbon dioxide is fixed into biologically usable carbon. The major player in the carbon fixation game is the enzyme RuBisCO. RuBisCO takes a 5 carbon molecule and attaches carbon dioxide creating a 6 carbon compound. This 6 carbon compound is then broken in half. RuBisCO can fix carbon dioxide but it can also use oxygen as well in a process called photorespiration. We will come back to photorespiration in a later post.

Calvin Cycle - personal illustration


The 3 carbon molecule generated by RuBisCo is called 3-phosphoglycerate. With a couple of enzymes, 3-phosphoglycerate is rearranged into glyceraldehyde 3-phosphate. This requires the usage of ATP and NADPH, both of which are generated in the light-dependent reactions.

The cycle part of the Calvin cycle is the many steps required to regenerate the initial 5 carbon molecule used by RuBisCo. A host of enzymes are required for the combination and rearrangement of carbon compounds to produce the initial 5 carbon compound (hence the many arrows on that part of the cycle). For each glucose molecule, the Calvin cycle has to turn 6 times using up 18 ATP, 12 NADPH, and 6 carbon dioxide.

Sum it Up

Photosynthesis is the real circle of life (sorry Lion King). Plants take carbon dioxide and fix it into glucose, while releasing oxygen as waste. Animals breath in oxygen, consume glucose, and release carbon dioxide as waste. Just another one of Nature's many recycling factories.

References

Falkowski and Raven, 2007. Aquatic Photosynthesis: Princeton University Press. ISBN: 9781400849727
Lodish et al., 2012. Molecular Cell Biology:W.H. Freeman & Company. ISBN: 9781464119132.

Vinyard DJ, Ananyev GM, Charles Dismukes G (2013) Photosystem II: The Reaction Center of Oxygenic Photosynthesis. Annual Review of Biochemistry 82 (1):577-606. doi:doi:10.1146/annurev-biochem-070511-100425