Saturday, April 9, 2016

Blog Update

Sorry for the lack of posts the last 2 weeks, hit a rough patch in real life. Between spring break work load and then child and I both got sick, there was no extra time to work on the blog. I made some goals for 2016 at the beginning of the year.

As a refresher the goals:
  • Read a scientific paper at least every work day, goal by year end 200 papers (#365papers"win" with 200)
  • Write more for the blog, one post a week (#50posts)
  • Shout out the little things that make me happy in Mommyhood (#PositiveParentMoment) 
  • 500 words a week in my PhD dissertation
  • Read 2 fun/non-sci books a month
  • Detailed edit of my NaNoWriMo 2015 by this upcoming NaNo  

Now the update...

What a hopeful person I was for this year! At this about 1/4 mark through 2016 I have
  • read 23 papers into #365papers before I got distracted and then fell behind and then forgot completely. 
  • written 12 blogposts(13 counting this one!) for #50posts (only 2 weeks off not terrible!)
  • #PositiveParentMoment has completely died off and I really need to refocus on that as it does help keep things with Boo in perspective
  • I have written 0, yes that's right, 0 words towards my PhD diss.. this needs to change STAT
  • I've read 3 fun books all year, short of the goal of 6
  • I did finish draft 1 of NaNo2015 and have started editing so I'll consider that goal on target
There we have it, out of 6 goals that I gave myself for 2016, I have sorta kept up with 2 of them.  Never to late to try again though! Here's to July 1 where hopefully I will have 100 papers in my #365papers Excel sheet, 26 #50posts, many thousands of words in PhD dissertation, and 12 fun books read!

We'll be back to science next week! 

Friday, March 18, 2016

Species Spotlight: Clover

As Saint Patrick's Day was yesterday, I thought we'd take a look at one of the popular symbols: the four-leafed clover. Botanically, clovers are any species within the Trifolium genus. They are in the Fabaceae family, as are soybeans! Over 300 species have been described within the Trifolium genus. There are clover species found practically worldwide. They make a very good cover crop and the most common honey is made from clover flowers which bees enjoy. Below is the common white clover (Trifolium repens).

The name trifolium literally means 3 leaves in Latin, and most clovers do indeed have only 3 leaves. But some clovers end up with 4 leaves. And these are the ones celebrated to give the finder good luck. Why are they so hard to find? To understand that we have to take a look at their genetics.. the blueprint that makes up life.

The genome, the entirety of the blueprints that makes up the organism, is pretty unique in this case. White clover has an allotetraploid genome, it has 1 copy of 2 different genomes! Each one of those genomes are diploid (2), this it has 4 gene copies! How does this happen? Well, in this case it is suspected that Trifolium occidentale and T. pallescens crossed. The resulting offspring kept both genomes to become T. repens. Because of this it has been difficult to map the genome and tease apart which genes control which traits.

Thankfully, there have been a few studies that have shed some light on this subject. It turns out that the progenitor of the Trifolium genus had 5 leaflets and the number of leaflets has decreased over evolutionary time to become 3 leaflets. But the 3+ leaflet is still a genetic possibility.  At least one multifoliate gene has been discovered. This gene is a recessive gene that is not only inherited (about 1 out of every 10,000) but can be activated by environmental conditions. Four leaf clovers like it hot, studies have shown there are more 4 leaf clovers in the summer than winter, and more in the greenhouse than outside during winter.

As Saint Patrick's Day occurs in March, this is not the ideal time to be searching for that "lucky" four leaf clover. Come back in June, when the days are warmer, to increase your odds!


Ford and Claydon Inheritance of multifoliate leaves in white clover. Agronomy Society of New Zealand Special Publication No 11. 167-170
Griffiths et al 2013. An integrated genetic linkage map for white clover (Trifolium repens L.) with alignment to Medicago. BMC Genomics 14:388
Gustine and Huff 1998. Genetic variation within among White Clover populations from Managed Permanent Patures of Northeastern USA. Crop Science 39:524-530
Tashiro et al 2010 Leaf Trait Coloration in White Clover and Molecular Mapping of the Red Midrib and Leaflet Number Traits.  Crop Science 50:1260-1268

Saturday, March 12, 2016

Species Spotlight: Crocus (with a little bonus Best Biochemist)

We are barreling towards spring here in the Northern Hemisphere. One of the early signs of spring are crocus flowers. The Crocus genus contains about 100 species. The flowers are cone shaped and live for about 3-4 days while they produce a massive amount of pollen. It's estimated that 1 flower can produce 1.9 milligrams (that's almost 7 ounces) of pollen!
Crocus from grandparents yard - personal image
Crocus often start to bloom in late winter/early spring. They can break through the snow. The flowers are, on average, 3 degrees Celsius higher than surrounding ambient temperature. The bowl shape of the flowers helps keep the heat in the interior floral area. Because they flower early, produce a lot of pollen, and generate heat in the flowers they are crucial for bee survival.

If you have any saffron in your house, you have a container of Crocus sativus stamen (flower male sex organs). Saffron is the most expensive spice in the world. It takes 70,000 hand-picked flowers stripped of their stamens to make 0.45 kilogram (about 1 pound). The scent, flavor, and color of saffron comes from unique carotenoids that are produced only by C. sativus. Technically, these compounds are apocarotenoids as they are shorter chains of carbon cut from zeaxanthin (which is also important in the xanthophyll cycle). The 3 apocarotenoids produced in the stamens of C. sativus are crocin, responsible for the red color, picrocrocin, providing the bitter taste, and safranal resulting in the characteristic aroma.

Researchers have been trying to elucidate the pathway used to produce saffron in the hopes of being able to synthesize it. Recently, the enzyme responsible for the initial step in this process was discovered to be a member of the carotenoid cleavage dioxygenase enzyme family.  All CCD are rigid 7-bladed propeller shaped with a central iron active site surrounded by 4 histidine residues which facilitate oxygen induced cleavage (cuts) of double bonds in carotenoids. CCD2 is the first enzyme to be described that is unique to saffron synthesis. CCD2 cuts zeaxanthin at the 7,8 double bond on both ends of zeaxanthin. Each bond is cut individually in a two step process to produce crocetin dialdehyde which is further processed to produce crocin, picrocrocin, and safranal.

It's amazing how one little flower can be so important and potent!


Frusciante, et al (2014) Novel carotenoid cleavage dioxygenase catalyzes the first dedicated steps in saffron crocin biosynthesis. Proceedings of the National Academy of Sciences of the United States of America 111 (33):12246-12251. doi:10.1073/pnas.1404629111 

McKee and Richards 1998 Effect of flower structure and flower colour on intrafloral warming and pollen germination and pollen-tube growth in winter flowering Crocus L. (Iridaceae) Botanical Journal of the Linnean Society 128: 369-384

Weryszko-Chmielewska and Chwil, 2011. Structure of the Floral Parts of Crocus vernus (L.) Hill Acta Agrobotanica 64(4):35-46
Whitney and Chittka 2007 Warm flowers, happy pollinators Biologist 54(3) 154-160.

Friday, March 4, 2016

In the News: Soybean Cold Stress!

In the Midwest US it is not uncommon to have a frost or snow event in early spring. My Mom's gardening practice is wait until after Mother's Day to be sure a cold snap won't kill her plants. Soybean are sensitive to cold, especially in the young seedling stage.  But why is this? And is there anything that can be done about it? Last week an important paper came out that shines some light on this subject.

Impact of cold on soybean seedlings (Pers. Photo)

The article looked at the CBF cold responsive pathway, which is known to play a very important role in cold acclimation and tolerance in other plants. This multi-step pathway is well documented in cold tolerant species, especially the labrat plant Arabidopsis. In this pathway, CBF (also called DREB, as it is in this paper) is the main transcription factor. A transcription factor is the cell's version of a foreman, going into the nucleus and telling the cell what genes need to be activated.

Cold stress in plants, personal illustration
Turns out, soybean also has these same genes. And that CBF turns on when soybean is exposed to cold much in the same way that it does in Arabidopsis. The researchers were able to draw a line in the sand between working and non-working portions of the CBF cold responsive pathway within soybean. Turns out only half the pathway is non-functional, the other half is working just fine! You might wonder how they figured that out, great question!

The first question that they examined was, does soybean have similar genes to Arabidopsis CBF cold pathway? By examining the soybean genome, they were able to compare gene sequences with the Arabidopsis genome to generate a list of potential homologs (genes that are the same between species). This is only good on paper, just because the sequences match does not guarantee the functions match.

But once you narrow an entire genome down to less than a dozen genes, it is easy to examine functional responses. Toss the soybean in the cold, use quantitative PCR to get a measure of the gene expression amount before and during the cold. When the authors did this, they found that the CBF transcripts (mRNA) increased drastically and transiently. This is the same response that is seen in Arabidopsis. From this they concluded that all the steps in the cold response are active in soybean, and the "problem" lies between CBF transcription and cold responsive gene translation.

Perhaps the "problem" is that CBF itself has mutated in soybean and is damaged so that it can no longer interact and activate the cold responsive genes. The researchers set out to test this by taking the soybean CBF and moving them into Arabidopsis. This new genetically modified Arabidopsis will always be expression the soybean CBFs. If soybean CBF works, it should turn on the Arabidopsis cold responsive genes without having to expose the Arabidopsis plants to cold.

Simplified Fig 5 from Yamasaki & Randall 2015, provided by S. Randall
And that is exactly what happened! The downstream cold responsive genes COR47, RD29a, and ADH1 all increased in the transformed Arabidopsis. This told the researchers that the soybean CBF are indeed functional, at least they are capable of activating their target genes when expressed in Arabidopsis.

So what is the "problem" with soybean's cold responsive pathway? We still do not know. But we do know that it is not upstream of CBF transcription. Thus this article allows us to focus in on part of the pathway as the "problem" area. Hopefully these researchers are still examining this and will find the solution!

Yamasaki, Y, and Randall SK, 2015.  Functionality of soybean CBF/DREB1 transcription factors (paywall)

Saturday, February 27, 2016

Best Biochemist: ABA

Growth hormones are critical to plant development, much in the same way they are for animals. Previously, I wrote about ethylene, the ripening hormone. Today, we'll look at abscisic acid (ABA), an important growth and stress hormone.

Biochemically, ABA is a 15 carbon derivative of zeaxanthin, a carotenoid, with the following chemical structure.

public domain
ABA was discovered over 50 years ago in abscising leaves. Abscission is the process by which plants detach leaves or ripened fruit. Thus the name abscisic acid. Though we now know that ABA does not play a role in abscission itself but rather cessation of growth that precedes the actual detachment process. In the 50 years since discovery, ABA has been shown to play a role in a multitude of pathways. These include: seed dormancy, embryo development, germination, cell division, flower induction, stress responses (drought, salt, cold, UV, pathogens), and stomatal closure. Let's start at the beginning of the plant's life cycle and take a look at the roles ABA plays in these processes.

Seeds in the ground remain dormant, in a state of suspended growth. The production of ABA is high during late embryo maturation which drives dormancy. Dormancy must be broken for the seed to germinate. When it is time for the seed to germinate, gibberellic acid and ethylene production results in a cascade that mutes the detection of ABA breaking dormancy. Germination then occurs and the plant grows.

As plants grow they can encounter many abiotic stressors. Personally, I research abiotic stress biology so I find the stress responsive role ABA plays to be one of the most interesting. Plant perception of environmental conditions such as, high salt, low water, cold, and wounding, all involve ABA. These stressors increase ABA levels which in turn activates stress responsive genes. The main function of ABA in all of these stresses seems to be controlling the water level within the plant in a number of ways, including gene activation, increasing water uptake in the roots, and closing the stomata.
Stomata Personal Image
Stomata are pores on the leaf that allow gas exchange, including water vapor. To close the stomata, ABA binds to receptors on the guard cells that control opening and closing of the stomata. These receptors allow the flow of ions such that the guard cells lose water and close the pores. This prevents water from leaving the leaf!

Flowering is an important stage in plant development, resulting in the production of seeds and the next generation of the species. As with all developmental phases, several hormones play important roles. The ratio of each and amount of cross-talk between the various hormone pathways results in the change of developmental stage.  Floral development requires an increase in ABA and decrease in ethylene to the proper ratio.

As the plants age, they will inevitably undergo senescence and die. The biosynthesis of ABA, along with a suite of other hormones, is increased while cytokinin is decreased. ABA works in a positive feedback manner. As ABA levels increase, water and minerals are pushed out of the leaf and into the stem. This results in dehydration of the leaf and further production of ABA. This sets up the leaf for abscission which is controlled by ethylene.

As we can see, ABA is an incredibly important hormone. It plays a role in almost every developmental stage a plant undergoes from germination to senescence. How exactly the plant knows when ABA means flowering, dehydration stress, or senescence is still a mystery. It is probably in part to genetic control, age of the plant, and coordination with other hormone levels. Plant growth and development is just as complicated as it is in animals.

Beaudoin, N., et al., 2000. Interactions between Abscisic Acid and Ethylene Signaling Cascades. The Plant Cell 12:7 1103-1115

Daszkowska-Golec, A., 2013. Open or Close the Gate - Stomata Action Under the Control of Phytohormones in Drought Stress Condition. Frontiers in Plant Science 4 138 doi 10.3389/fpls.2013.00138

Finkelstein, R. 2013. Abscisic Acid Synthesis and Response. Arabidopsis Book. 2013; 11: e0166. Published online 2013 Nov 1. doi:  10.1199/tab.0166

Lee, I., et al, 2011. Age-dependent action of an ABA-inducible receptor kinase, RPK1, as a positive regulator of senescence in Arabidopsis leaves. Plant Cell Physiology 52(4) 651-662

Tuteja, N. Abscisic Acid and Abiotic Stress Signaling. Plant Signaling & Behavior 2(3) 135-138 

Wilmowicz, E., 2008. Ethylene and ABA interactions in the regulation of flower induction in Pharbitis nil. Journal of Plant Physiology 165 (18) 1917-1928

Sunday, February 21, 2016

Minecraft Character Ornaments

This week my laptop got stolen and I lost my science post I was working on, which was very disheartening. So I'm going to take a short break from my normal science-y posts and bring you this post about Boo's Minecraft birthday party from a few months back!

One of the activities I came up with was making a Minecraft ornament. I had found some 1.5 inch paper mache block ornaments from Micheal's at 1/2 off so they were < $1 each!

To simplify things during the party, I created 4 color packs with 3 face options they could copy onto their blocks. Since the ornaments had 4 sides, the kids would rotate around the packs picking a face and gluing the correct color onto the ornament base. Then they would leave with an ornament full of their favorite Minecraft faces!

The packs were:
Green - Creeper, Zombie, Slime
Black - Enderman, Spider, Ender Dragon
Tan - Steve, Alex, Sheep
Gray - Skeleton, Wolf, Moo-shroom
After the party, for fun I also created a squid and ocelot pattern.

After I designed the patterns, I cut 1/2 inch squares out of all of the colors I needed from scrapbook paper. I had a dark green, light green, black, purple, pink, red, gray, tan, brown, white, orange, yellow, and blue.

A few characters required special pieces. For example, the creepers mouth, slime mouth, zombie eyes, and squid pupils. I cut all of those out by hand.

 The paper squares were put into plastic cups as needed for each station. Once the kids got started, they really enjoyed this activity! I gave each boy a glue stick for them to glue each piece. Some decided to drag the paper square over the glue then put it on, others spread glue all over the ornament and then stuck the paper onto the glue.  

On the bottom, I added the logo from Boo's party with the date so they would know where they made it. On the top, I had them put their names so while the glue was drying and we were doing our scavenger hunt we would know which ornament went in which goodie bag!

This project was a lot of fun. Since I have a big paper cutter, it was easy to set up as well. Finished ornaments were adorable and the boys raved about them. You can see some of the resulting faces below!

 This Friday we'll be back to science, thanks for indulging me on my Mom post!

Sunday, February 14, 2016

A Valentine's Day Species Spotlight!

Roses are Red
Violets are Blue
Sugar is Sweet
And so are you! 

A classic oldie, but goodie poem that makes the rounds on Valentine's Day. For this very special Species Spotlight we are going to take a quick look at the 3 plants that star in this classic nursery rhyme.

Personal Image

are not always red! I briefly talked about roses at July 4 as they are the national flower of the USA. The Rosaceae family contains over 2500 species, including some agriculturally important ones: apples, strawberries, raspberries, pears, plums, and more. The poem above is, of course, talking about flowers from the genus Rosa. There are over 100 species in this genus.

And while "Every rose has its thorns" is a popular lyric, rose thorns are not true botanical thorns but rather prickles. Thorns are a modified branch. Prickles are modified from the epidermis tissue on the stem. Though I suppose "every rose has its prickles" does not sound as sharp ;)

Personal Images

are not usually blue! The Violaceae family contains around 800 species, the commonly called violet flowers are within the genus Viola. Viola sororia or the common blue violet, is quite popular representing Illinois, Rhode Island, New Jersey, and Wisconsin as the state flower. Violets have 5 petals and 5 sepals (the green part behind the petals) that are arranged asymmetrically. Their leaves and petals can be heart shaped which makes them a nice match for Valentine's Day.

The bright coloration of violets is to attract pollinators, such as bees. The bees will have to burrow deep into the flower to get to the nectar, ensuring pollen will be transferred to the bee to be taken to another violet. Violets have fascinating seed pods that allow the seeds to become flying projectiles. Drying out of the seed pod increases the pressure and eventually POP! Seeds go flying. This allows the violet to spread rapidly. This rapid spreading, and hardiness of the seeds, contributes to the ability of violets to quickly spread over an area. Watch the video below to see the amazing exploding violet seed:

Another incredible way that violets can spread seed is via ants. Violet seeds are covered in a rich oily covering called an elaiosome. This sugary oil attracts the ants which drag the seed back to their nest. After the elaiosome is consumed, the exposed seed can now germinate in ant fertilized (read full of ant poop) soil. This mutulistic relationship has a very long fancy name: myrmecochory and it is found in 1000s of other flowering species.

Sugar Cane

is indeed sweet, after processing. Sugar cane is a grass from the genus Saccharum and it is in the same family as other important crops such as wheat and rice. Sugar cane is though to have been introduced to America by Christopher Columbus. The tall, thick stems of sugar cane have a sucrose filled sap. This sap is removed and then boiled and crystallized to form sugar! Since the stems, and not the fruit, are the agriculturally important part, they can be harvested repeatedly without having to replant from seed.

As a fan of photosynthesis, in my opinion one of the coolest things about sugarcane is how they do photosynthesis. They do what is known as C4 carbon fixation. Light capture happens the same way in C3 ("normal" photosynthesis) and C4 plants, it is the Calvin Cycle that is different. One of the downfalls to photosynthesis is photorespiration, when RuBisCo utilizes oxygen instead of carbon dioxide. To get around this, C4 plants have a special modification, called kranz anatomy.

Inside the leaf, there are "wreath" shaped bundles of bundle sheath cells surrounded by mesopyll. Only the bundle sheath cells have RuBisCo and all of the carbon dioxide produced by the light-reactions is passed to these bundle sheath cells from the mesophyll cells. The movement of carbon dioxide between the cells is facilitated by a 4 carbon compound, hence the name C4 carbon fixation. This is advantageous as it concentrates the carbon dioxide around RuBisCo and drastically reduces the amount of photorespiration. Reducing photorespiration means that more energy goes into sugar production. Thus C4 carbon fixation is the true reason sugar is so sweet.

And so are You

Happy Valentine's Day to all of my readers!


Friday, February 5, 2016

Species Spotlight: Garlic Mustard

One of my favorite plants is Garlic mustard, or as it is known in science Alliaria petiolata. It was the first plant I spent a significant amount of time researching, forming the backbone of my honors undergrad thesis. This is why, despite its reputation, I will always have a fond spot for this weed. 

2nd year Garlic Mustard - Personal image
Garlic mustard is a biennial mustard native to Europe. In 1868, the first documented garlic mustard arrived in New York and thus begins an alien invasion. Genetics has suggested that garlic mustard was brought over multiple times, from multiple regions in Europe which has led to a high genetic diversity in North America.

As a biennial, garlic mustard requires 2 years to flower and produce seed. The first year is spent as a rosette, a small bundle of leaves.In the second year, the inflorescence (flowering stem) shoots up. Each plant can produce thousands of seeds. Each seed can live up to 5 years in the soil before germinating. The longevity of the seed bank is one of the reasons garlic mustard is difficult to control. It would take over 5 years of gathering every single one of those thousands and thousands of  plants that germinated from the seed bank to successfully clear an invaded area. 

Garlic Mustard with siliques (seed pods) - Personal image
Garlic mustard is able to wage chemical warfare upon other seedlings and mycorrhizal fungi. Mycorrhizal fungi is symbiotic with the roots of many plant species, they provide nutrients that the plants are unable to synthesize and vice versa. When garlic mustard invades a region the mycorrhizal fungi is greatly reduced, resulting in the death of the native plants that rely on it. For garlic mustard this is perfect, more real estate! For the invaded forest this decreases the species diversity.

As the name suggests, garlic mustard tastes like a mix of garlic and mustard. This particular  combination is disliked by the common large herbivore in North America: deer. Deer ignore garlic mustard, but eat the native plants. Clearing even more room for garlic mustard to invade!

Me taking field notes in a field of garlic mustard (white flowers)
Lots of factors have played into garlic mustard's takeover of North America, yet it is not preset across the entire continent. One of the factors that has limited garlic mustard spread is it requires a lengthy cold period for germination. The mimicking of a natural cold period in the lab is called startification. During startification, the seeds are kept moist and cold, mimicking the conditions of cold/frozen ground. This softens the seed coat so that when temperatures increase in the spring the seed germinates. Garlic mustard requires 12 - 16 weeks of startification for germination. This is what keeps garlic mustard contained to Canada and the northern US. Beyond this, there are not many other road blocks to stop this alien invasion.

The most effective control method is pulling the plants before they can make seeds. For small invasions this works well. However, garlic mustard's fast spreading rate (some predictions say >3000 miles/year) and the fact that seeds can persist for 5 years makes manual removal a difficult task for larger invasion areas. Herbicides are effective but can cause further damage to native flora. 

If you find some garlic mustard, pull it before it makes seeds. If you are feeling adventurous, you can use the plants for a number of recipes! My undergrad advisor had a soup recipe that was pretty tasty! A number of which have been put together in the Mid-Atlantic Invasive Plant Council Garlic Mustard recipe file. I haven't had the chance to try these recipe but that ricotta dip will be a must try this spring! Garlic mustard should be coming up in March/April so let me know which of the MAIPC recipes you try!

Delaware DNR made an info video about garlic mustard which I will leave you with:

  • Baskin, J. M., C. C. Baskin. 1992. Seed germination biology of the weedy biennial Alliaria petiolata. Natural Areas Journal 12(4):191-197.
  • Durka, W., Bossdorf, O., Prati, D., & Auge, H. (2005). Molecular evidence for multiple introductions of garlic mustard (Alliaria petiolata, Brassicaceae) to North America. Molecular ecology, 14(6), 1697-1706. Research Gate PDF
  •  Meekins, J. F., and B. C. McCarthy. 1999. Competitive ability of Alliara petiolata (garlic mustard, Brassicaceae), an invasive nonindigenous forest herb. International Journal of Plant Sciences 160(4):741-752.
  • Nuzzo, V. 1991. Distribution and spread of the invasive biennial Alliaria petiolata (garlic mustard) in North America. P. 137-145. In McKnight, Bill N. ed. Biological Pollution: The Control and Impact of Invasive Exotic Species. Indiana Academy of Sciences: Indiana, USA. 
  • Rodgers, V. L., Stinson, K. A., & Finzi, A. C. (2008). Ready or not, garlic mustard is moving in: Alliaria petiolata as a member of eastern North American forests. Bioscience, 58(5), 426-436. 
  • Wolfe, B. E., Rodgers, V. L., Stinson, K. A., & Pringle, A. (2008). The invasive plant Alliaria petiolata (garlic mustard) inhibits ectomycorrhizal fungi in its introduced range. Journal of Ecology, 96(4), 777-783. PDF

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.

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.

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