Category Archives: journal club

The Dangerous Double Life of a Distinctive Diazotroph

For many people around the world, tonight is one of the most anticipated nights of the year. In that spirit, here’s some new science, not under the sun, but under the moonlight and starlight. New research from Wegener, Nagarajan and Pakrasi* describe something new about the Photosystem II D1 protein, an unexpected source that photosynthesis researchers had long written off as conquered or fully explained.

Why would we care about photosynthesis at night anyway? It all comes down to the distinctive lifestyle of the cyanobacterium Cyanothece, which belongs to an interesting class called unicellular diazotrophs (word of the day). Like all cyanobacteria, they perform oxygenic photosynthesis but they have the bonus biochemical ability of nitrogen-fixation.

Nitrogen-fixers are capable of pulling nitrogen gas (N2) out of the air and turning it into ammonia which the cells can use as a nitrogen source for important molecules like protein and DNA. Other microbes can perform nitrogen-fixation, and you may remember them as the root nodule symbionts of legumes. If you’re not a nitrogen-fixer, you have to rely on ammonia or other nitrate compounds present in the soil, sea or your diet (depending on what kind of organism you are) to make the required nitrogenous biomolecules. Since the Earth’s atmosphere is 78% N2, you don’t have to be great at math to figure out that acquiring the ability to tap into the atmospheric nitrogen source advances your species in the game of life a considerable number of squares.

What does this have to do with photosynthesis? Why don’t more plants and cyanobacteria get in on this nitrogen-fixation biochemistry? There’s a critical fatal flaw in the photosynthesis/nitrogen-fixation biochemical tango. Nitrogenase, the enzyme responsible for the nitrogen-fixation reaction is hopelessly and irreversibly killed by oxygen. Oxygen destroys the protein enzyme that’s already been made and tells the genome not to even bother wasting energy trying to make it new. If you’ve been paying attention to the blog, oxygenic photosynthesizers like cyanobacteria and plants make oxygen via their PSII enzyme every time light shines on them. So for nonphotosynthetic soil bacteria and other microbes, it’s not a big deal to avoid oxygen and go anaerobic to fix nitrogen. Cyanobacteria, however, must lead dangerous double lives to combine these two opposing biochemical processes.

Anabeana spherica via Wikimedia Commons The more round cell in the center of the string is the nitrogen-fixing heterocyst.

Some diazotrophic cyanobacteria solve this problem by separating these two processes in space. Species like Anabeana can go through a special type of development where the cells divide themselves, but incompletely to form a connected network like beads on a string. Every tenth cell (heterocyst) gets an extra layer of cell wall, destroys its photosynthetic machinery, and commits itself to a life of nitrogen-fixation. These cells share their fixed nitrogen compounds with their neighbors, which in turn share their fixed-carbon from photosynthesis. This is a fascinating process still hasn’t given up all of its secrets and remains an active area of research.

Cyanothece 51142 by Michelle Liberton, Pakrasi lab

Cyanothece 51142 by Michelle Liberton, Pakrasi lab

Cyanothece, on the other hand, is a more ruggedly individualistic species that manages to perform both oxygenic photosynthesis and nitrogen-fixation within a single cell. It accomplishes this through a carefully controlled circadian rhythm. During the day, oxygenic photosynthesis occurs. During the night, nitrogen-fixation occurs. It sounds so simple, yet this elegance is far from easy. This is a true circadian process, in that, while some cues are definitely derived from light signals, the cells also have a strong internal clock that keeps the time of day and controls what biochemical machinery is active. Thus, near the hour of dawn and dusk, the cells are actively preparing for the metabolism to come. Also, if you were to train a culture of these cells under a certain day/night cycle for several days then switch them to continuous light, they would still maintain their circadian biochemical cycle for several days after the switch.

One issue that has puzzled researchers for some time is that the photosynthetic machinery, like PSII, is actively shut down at night. It’s not just that it’s dark, so there won’t be any photosynthetic oxygen production. Apparently, that’s not good enough. The cells actively shut it down. When researchers take culture samples of these cells during their dark period and shine light on them to check photosynthetic capability, there is no signal. Sure, there’s probably little chance any significant photosynthesis is occurring at night, but Cyanothece isn’t taking any chances. For these cells, no moonlight, floodlights from passing ships, comets, or even the blessed Eastern Star is going to interfere with their nocturnal nitrogen fixation.

Shutting down photosynthesis is no small feat. Again, if you’ve been paying attention to the blog, you will note that I’ve ranted on numerous occasions about how complex PSII is and how difficult it is to assemble  and reassemble from its individual parts. As far as energetics are concerned, it would be a losing battle for Cyanothece to completely disassemble its PSII at dusk only to resynthesize it again the next morning. I don’t care how hard-up it is for a nitrogen source. As it turns out, Cyanothece and other unicellular diazotrophic cyanobacteria solve this problem in a more graceful way, for which Wegener et al have provided new experimental evidence.

Cyanothece, like all other oxygenic photosynthesizers, contains multiple copies of the psbA gene encoding the D1 protein, which is a core subunit of PSII and critical for function. Since the advent of the genomic era, the genomes of numerous photosynthetic organisms have been sequenced revealing these multiple psbA genes, most of which have little or no protein sequence differences. These subtle changes have been linked to cells’ ability to fine-tune photosynthesis under certain conditions. However, Cyanothece and its cousins have a psbA gene copy that is particularly different- psbA4. If one were to take all of the site-directed mutants that photosynthesis researchers made to sort out PSII function over the last few decades since molecular biology techniques were available and threw them altogether into a single mutant gene, you would get psbA4. If PSII contained the D1 protein encoded by psbA4, there would be no C-terminal processing and no active site for water-splitting and oxygen production.

Why would any self-respecting photosynthetic organism retain such an utterly non-functional PSII subunit? The answer is because it does serve a critical function in the daily cycle of Cyanothece, just not during the day. The D1 protein encoded by the psbA4 gene, called a sentinel D1 protein (sD1), is made only for the night, when it does the only thing it still can do- fit right in the center of the PSII complex and prevent PSII function of any kind. There’s no chance of any oxygen production while sentinel D1 is on duty, but the cells can maintain most of the other PSII components in a stable complex (meaning they don’t have to be degraded and made fresh every morning). Then, Cyanothece can turn photosynthesis back on in the morning by replacing a single protein in its PSII complexes. The report by Wegener et al gives the first biochemical characterization of PSII complexes containing a sentinel D1 protein, providing evidence that this strategy works for controlling PSII oxygen production.

sentinel

Performing oxygenic photosynthesis and nitrogen-fixation within the same cell is biochemically problematic, but the sentinel D1 protein appears to be the secret weapon for this dangerous double life. Through this strategy oxygenic photosynthesis and nitrogen fixation are “Always together, eternally apart.”** Many questions remain regarding the regulation of the transition from functional D1 to sentinel D1 at dusk and the reverse replacement at dawn. There are still outstanding questions regarding the D1 turnover event for any PSII in any photosynthetic organism, so it’s not at all clear what parts of this process Cyanothece uses or whether new factors are involved. Obviously, there must also be some sophisticated regulation to prevent the synthesis of sentinel D1 during the day.

While this work focuses on an organism you’ve never heard of, it has some interesting biotech implications for the future. As I mentioned, being able to both fix CO2 through photosynthesis and N2 through nitrogenase puts you at a significant metabolic advantage. And no, I don’t mean you. Remember, we’ve been through this- people are not photosynthetic. It would be great if one day we could engineer certain crop plants to combine these traits. Legumes already have this trick covered through symbiosis, but many other staple crops require tons of nitrogen fertilizer. If researchers could unlock the secrets of Cyanothece’s double life and translate it to plants, then it may be possible to engineer versions of crop plants that don’t require so much nitrogen fertilizer. Thus, one night in the future, engineered crop plants may be fixing nitrogen thanks to the addition of a variant gene they already had.

Johnna

*I know I’ve posted frequently about Pakrasi lab (my thesis lab) research, but this topic was just developing as I was leaving the lab and it’s too cool to pass up.

**Bonus points to you if you get the movie reference.

References and Links:

http://www.jbc.org/content/early/2014/12/18/jbc.M114.604124.full.pdf+html?sid=8f38f7c6-96c1-4085-bbb0-efc6ca425624

Two Tales of a Manuscript

It has been a shamefully long time since I’ve done a post for the Journal Club category. So today’s will be a deluxe edition of Dickens-proportions. Normally, you only get the science tale as presented in any journal article, neatly fit to the scientific method. However, for every scientific publication, there is another tale, a more elaborate backstory with twists, turns and subplots. While these secondary tales may be more dramatic, the traditional publication process relegates them to the shelf locked inside lab notebooks. Well today you will be getting both tales, because I’ll be breaking down my latest accepted manuscript. Read the science version (Tale 1), the behind-the-science version (Tale 2) or both.

I’ll be enlisting the help of Charles Dickens because many of the quotes from A Tale of Two Cities are as true for the practice of science as they are for complicated human struggles with relationships, sacrifice and revolution.

Tale 1

“It was the best of times, it was the worst of times.” Charles Dickens

All science projects seem this way. They can begin so full of promise, then change direction down a pathway you were not expecting and perhaps did not want to follow. You may think you know what you are doing, but some results case doubts. You have come to a conclusion, but then do one experiment too many and it shatters.

The PsbP-Domain Protein 1 (PPD1) Functions in the Assembly of Lumenal Domains in Photosystem I

Hypothesis: The lumenal protein PPD1 plays a critical role in photosynthesis, specifically in the accumulation of Photosystem I (PSI)

Experiments: In the model plant Arabidopsis, RNAi mutants of the PPD1 gene were characterized with respect to photosynthetic activity. The RNAi technique allows researchers to target a specific gene and knockdown its expression quickly and easily. These mutants can show a range of phenotypes that are useful in teasing apart the functions of genes whose complete elimination causes the death of the organism. The PSI activity in the PPD1 RNAi plants was extensively characterized as was the accumulation of many thylakoid membrane proteins (including PSI subunits). Native gel electrophoresis was also used to characterize the state of thylakoid membrane protein complexes in wild-type and PPD1 RNAi mutant plants.

PSI activity in PPD1 RNAi plants and representative plants from each group I-IV

PSI activity in PPD1 RNAi plants and representative plants from each group I-IV

Results: The PPD1 RNAi mutants with the lowest PPD1 expression were extremely small and pale green plants. Analysis of chlorophyll fluorescence showed that the mutants had much higher levels of fluorescence, indicative of an over-reduced plastoquinone pool and problems on the PSI-side of the photosynthetic electron transfer chain. Specific measurements of PSI activity showed that the PPD1 RNAi mutants had reduced amounts of active PSI reaction centers. However, energy could be transferred to these reaction centers by an alternative antenna system (LHCII). Moreover, the function of the PSI centers which did accumulate was not normal. Further analysis of protein accumulation in the thylakoids of the PPD1 RNAi mutants revealed there were specific problems in the accumulation of proteins on the lumenal side of PSI. In wild-type plants, the PPD1 protein was found to be associated with a thylakoid protein complex of ~300 kDa, which is smaller than any PSI complex.

2D gels showing thylakoid protein complexes in WT and PPD1 RNAi mutant. 1,2,4 complexes are forms of PSI; 3 is ATP synthase

2D gels showing thylakoid protein complexes in WT and PPD1 RNAi mutant. 1,2,4 complexes are forms of PSI; 3 is ATP synthase

Conclusions: The PPD1 functions in the proper assembly of PSI components on the lumenal side of the complex. In this area, PSI contains an extrinsically associated protein, PsaN, as well as extensive loop regions of the membrane proteins PsaA, PsaB and PsaF. All of these components create the binding environment for the soluble electron carrier, plastocyanin, which delivers electrons from upstream in the transfer chain. Reduced amounts of PPD1 affect the accumulation and assembly of these components. The mutant plants try to compensate for this loss of functional PSI by shifting some of the LHCII antenna such that it can funnel energy into PSI (the default for LHCII is to drive energy into PSII). The PPD1 protein is not considered a subunit because it was not found to be associated with fully assembled PSI complexes, but a smaller protein complex.

Think Ahead: The assembly of PSI is not a well-characterized biological process because, unlike PSII, PSI is an extremely stable enzyme. Thus, once it is assembled, the complexes can function properly for very long periods of time. Because it is a rare process, it is difficult to study. The original characterizations of PSI subunit mutants were performed many years ago, and it may be interesting to give them a fresh look with respect to PPD1 and some of the antenna effects we described. Identification of the other proteins in the complex with PPD1 (other PSI subunits perhaps) may help to define a PSI assembly intermediate. The secondary effect that the PPD1 mutation had on the antenna system will also be interesting to follow-up on because we don’t really know all of the details governing how plants allocate light energy between the photosystems. It is a sophisticated system with multiple layers of control.

 

Tale 2

“Nothing that we do, is done in vain. I believe, with all my soul, that we shall see triumph.” Charles Dickens

While scientific endeavors may have their dark moments, scientists tend to think that ultimately their research will see triumph. In the world of academia, this means publication in a peer-reviewed journal. Thus, all of the experiments that were done leading up to that publication but not included in it are not done in vain. They helped to work out the procedures necessary for acquiring the data that did appear in the figures. They were experiments that yielded negative data which eliminated hypotheses. Alas, those are never published.* It may be useful for scientists to know what wasn’t, but publishers only want to tell the stories of what was. (Hey, that almost sounds like Dickens too.)

The PPD1 story started with a blanket search for functions of the PPD family of proteins in the thylakoid lumen. They must be doing something to help plants photosynthesize, right? I was hopeful that maybe one of them had something to do with my favorite enzyme PSII. The way I chose to attack this problem was to characterize mutants of each of these proteins in Arabidopsis.

The easiest way to acquire Arabidopsis mutants is to order T-DNA lines (insertion mutants) for your gene of interest from the ABRC stock center. They send you seeds and you check to see if the mutants are useful or show a phenotype. There were two available lines (independent insertions) for the PPD1 gene and both of them were less than helpful to me. One line that another postdoc had been working with that had been passed on to me, which may have shown a phenotype, turned out to be heterozygous (not a complete mutant; still contains one wild-type PPD1 gene and should be normal). I couldn’t replicate any subtle phenotype, but neither could I find any homozygous (complete mutant) plants. Ever. I spent a lot of time verifying that that particular T-DNA mutant was embryonic lethal for homozygotes. Strange, but possibly extremely interesting. However, before taking this as a fact, it had to be confirmed that the homozygous lethal phenotype was because of the mutation in the PPD1 gene and not some other random mutation elsewhere in the genome. This can be sorted out by backcrossing heterozygote mutants to wild-type plants a few times and trying to recover the mutants. Ultimately, my experiments showed that the link between the PPD1 mutation and the embryonic lethal phenotype was not so absolute. At the same time, I was growing the other T-DNA mutant and it was proving to be equally unstable. Some plants would show a variegated phenotype, streaked leaves with patches of green and pale yellow. Other plants looked normal. I could never consistently link the phenotype to the ppd1 mutant genotype. With all of these inconsistencies in results, I decided that the T-DNA mutants were not useful in telling me anything about PPD1 function.

The alternative approach to Arabidopsis mutants is to use the RNAi technique to selectively suppress the expression of your favorite gene. While I was wrestling with the PPD1 T-DNA lines, I began the process of generating my own PPD1 RNAi lines. These plants turned out to be the most useful for figuring out what PPD1 does. When screening through these mutant individuals, there was a range in what the individual plants looked like- some looked almost normal while others were very small and pale. This is the good thing about RNAi because this range among individuals gives so a sort of picture of what is happening when the expression of the gene of interest is turned up or down over a gradient, like tuning up a brightness or volume knob on an old TV.

There was very little material to work with for the most severely affected PPD1 RNAi plants, but the biophysical measurements I could do on the tiny leaves indicated there was no problem with PSII. The defect was further downstream, probably in PSI. When using an instrument to specifically measure PSI function it was clear that was where the problem was. I would have to learn more about the PSI complex to say enough to turn my results into a publication, but at least I knew where this was going.

 

“There is prodigious strength in sorrow and despair.” Charles Dickens

The same week as my PSI results, I received an after-hours e-mail from my PI with the link to the following journal article: PsbP-domain protein1, a nuclear-encoded thylakoid lumenal protein, is essential for photosystem I assembly in Arabidopsis, Liu et al 2012 Plant Cell. When I quickly skimmed the abstract, my heart sank. My response was $%&^!, $%^@!, #$%@!, *&$%! I think I even drowned my sorrow in a pint of Hagen-Daz. There was only the slightest glimmer of satisfaction from the validation that researchers on the other side of the globe had come to the same conclusion as me.

Validation is not the name of the game. You see there is no prize for second place in scientific publishing. When you are the first group to publish a new idea, you have more control over the limits of the tale are. When you are second place, you cannot merely confirm what has been done (PPD1 has something to do with PSI). You must take it further, press on to unravel more details. Pressing on into the details of PSI territory was not really what I wanted to do.

However, after carefully reading what Liu et al had done, I reassessed my data and found a way to move forward. They had managed to characterize a clean T-DNA line, and the homozygous mutant plants they worked with were completely devoid of PSI. The small pale plants had to be grown on sucrose-containing medium since they could not support themselves photosynthetically. In my work, the RNAi lines allowed me to characterize plants that were very sick, but could still grow on soil. They accumulated some PSI, which could be analyzed more closely. Of course, that meant that I had to do a lot of experiments on precious little material. These experiments meant using a lot of brute strength just to get enough material for the experiments (spectroscopy measurements and blots, oh the blots!), investing time in fine-tuning protocols and money in antibodies for our second-favorite thylakoid complex.

“Vengeance and retribution require a long time; it is the rule.” Charles Dickens

Pushing forward with the experiments was difficult and took quite a bit of time. The sickest of the PPD1 RNAi lines were very small and would not set seed. Getting enough material meant screening for primary transformants every time. Learning the literature for a different enzyme complex was challenging. The papers describing the original characterizations of PSI subunit mutants were at least a decade old and often lacked data I would have liked to have seen. Not really flaws with that work; it’s just that what was not necessary for that work would have been extremely helpful to me.

Eventually, all of my data was written up in manuscript form and submitted away for peer review. I dabbled in other projects waiting the weeks until reviews came back. It took longer than usual, which meant only one thing- it was sent to a third reviewer. Yes, the two reviewers that initially evaluated my work had such differing views as to what my manuscript’s fate should be, a third reviewer was enlisted to help the editor in making the appropriate decision. Please revise with additional experimentation and there were specific concerns about how we went about doing some of our experiments.

Yes, a long time is the rule. I spent the next months painstakingly addressing the reviewer’s points with new experiments. One issue was how we estimated the amount of PPD1 protein in the mutants. With our antibody and a number of variations of our gel system, the PPD1 protein ran at the same molecular weight as the LHC proteins- the most abundant membrane protein on earth. These proteins obscured the signal for PPD1 such that we could never reliably estimate its amounts on denaturing gels. It either could not be seen or samples would require too much handling and treatment to consistently give a signal. However, PPD1 could be perfectly detected on native gels because the LHCs were nowhere near it in that system. Finally, I had point-by-point addressed all of the issues, revised the manuscript and created new figures.

We were ready to try again, but the tone of one of the reviewer’s comments gave us pause about resubmitting. Sure we had responses, but the original comments seemed like they would never invite satisfaction. There were some things about our results that would just not change. Experiments were done properly and yes, the results were still slightly unexpected. We would not be making up data because it would be easier for reviewers to accept. That is a cardinal sin in science, and a separate rule that should never be broken. For the revised manuscript, we took the chance on submitting to a separate journal with different reviewers for the chance of a favorable decision. This wager did not pay off because the two new reviewers had a completely separate set of comments to be addressed, many of which seemed impossible to satisfy with our sample limitations. We declined the invitation to revise and resubmit and ultimately resubmitted to the original journal. It went back to the original reviewers who seemed mostly satisfied with the improvements. Of course, there were some new comments by the reviewers that we could just not accommodate; not because the concerns were invalid, but because the questions went well beyond the scope of our work. There will always be more experiments to be done, but we firmly and politely stated we would not be addressing the new questions our latest experimental results sparked. We could only speculate as to future possibilities in the discussion section.

“A multitude of people and yet solitude.” Charles Dickens

In the case of this particular project, it was a multitude of data, yet not figures. I have numerous notebooks filled with raw data from experiments related to PPD1. In science, you go through a lot of preliminary work to get the answer, but when you show your work it must be much neater (certain colors, intensities, certain samples in certain orders). It’s like taking a math test and having a separate scratch paper (the lab notebook), but your answer is only a single circled number or neat graph (the manuscript figures). In this particular case, it was a multitude of blots. I cannot tell you how many blots I developed for this project. I practically lived in the dark room for months, eagerly waiting for films to emerge from the developer, praying the signals would be beautiful enough for figures.**

“I wish you to know that you have been the last dream of my soul.” Charles Dickens

Appeasing the reviewers in this final round felt a lot like the emotions in this quote. I had started forming the publication framework haphazardly because it wasn’t on a topic that I found exciting. Admittedly, I was only trying to do just enough to get acceptance. Even though my sentiment for some of my reviewers was more akin to a different saga, their requests did make the story better and forced me to expand my general knowledge on PSI and technical expertise in new protocols. All research can continue ever and on, but lines must be drawn somewhere because of the universal limits of effort, time and finances. I felt that the story was finally new and good with enough potential tangents to drive future research by possibly myself and others in the field. I had finally come to the point that I didn’t just want it done for the sake of adding another publication to the tally, but I wanted it published because the results deserved to be part of our body of photosynthesis knowledge.***

“It is a far, far better thing that I do, than I have ever done; it is a far, far better rest that I go to than I have ever known.” Charles Dickens

My PPD1 manuscript was eventually accepted at the Journal of Biological Chemistry this summer after a very long road of experimental struggles and research-related drama. Of all of my publications, this was definitely the most difficult to get to the point of publication. It is probably at the bottom of the list of my works if I had to rank them my favoritism based on any scale. This post was actually quite difficult to write; I so long to leave it in the past. However, I can now recognize that it is one of the better things I have done just to not give up on it. And after all the co-authors thought that everyone who would ever be interested in the PPD1 protein would have written the paper or reviewed it, we get an e-mail from another research group requesting our PPD1 antibody because their work may have a link to PPD1. “We read your paper with great interest,” they said in an e-mail sent a mere two days after our accepted manuscript appeared on-line. “They did!” I laughed. I sent them a sample, some behind-the-science instructions and well wishes. Apparently, my perseverance wasn’t just a better thing in terms of racking up publication numbers on my CV, but also for some other researchers embroiled in their own scientific epic. The best of times and the worst of times indeed.

As for me, it is a far, far, better rest that I go to than I have ever known as well. It’s not just a project change and definitely not the guillotine. Announcement coming soon to the blog.

 

Johnna

*However, Elsevier is launching a new journal where you can publish those results. Introducing the new journal New Negatives in Plant Science.

**There is a popular song this summer by Lil Jon and LMFAO “Shots”. There are not many lyrics. The rapper mostly just says shots over and over the backbeat track. To stave off insanity, or perhaps the opposite, I would sing my own version of the song “Blots.” All my biochemists, where y’at? Let’s go. When I walk in the lab, gloves on me, with the antibodies, I love chemiluminescence, I came to develop, lights off, it’s on! Blots, blots, blots, blots, blots, blots, blots, blots, blots, PPD1, blots, blots, blots, blots, blots, PsaB, blots, blots, blots, blots, blots, blots, LDS-PAGE, blots, blots, blots, blots, blots, Blue native, blots, blots, blots, blots, blots… If I don’t do these blots, I can’t resubmit! You get the idea. For visual effect, you can also picture me making it rain with x-ray films. Hey, what do you know another parody for the blog.

***Although if it would not have been accepted, I had threatened to just dump all of my results on this blog anyway and be done with it. Not sure how it ranks in terms of impact factor though.

References and Links:

http://www.jbc.org/content/early/2014/07/09/jbc.M114.589085

https://www.goodreads.com/work/quotes/2956372-a-tale-of-two-cities

Adamantium, for plants!

Researchers have basically created the plant equivalent of Wolverine

Researchers have basically created the plant equivalent of Wolverine

One of the goals of photosynthesis research is to improve plants’ productivity to a level adequate for human food and energy needs. As of now, plants are currently doing a good enough job to reproduce themselves and mostly sustain our biosphere, but we would still like to kick this up a notch. Scientists are using a variety of tools to tackle this problem. The most notable is using genetics- either by breeding new varieties or genetically engineering new traits by manipulating specific genes sometimes between different organisms. Today’s post features another type of engineering to improve plants ability to photosynthesize- nanotechnology.

Researchers at MIT report that plant chloroplasts can be infused with carbon nanotubes to impart increased photosynthetic capacity (as much as 49% greater than unaltered chloroplasts and 30% in whole plants). Much like the X-Men character Wolverine was infused with the metal alloy adamantium to render his skeleton indestructible and give him those awesome retractable claws, plant chloroplasts can be infused with special materials to give them what amounts to photosynthetic superpowers. No, they didn’t use adamantium. The material used in their study was carbon nanotubes fused to cerium oxide nanoparticles aka nanoceria.*

You may be wondering to yourself, “What exactly are photosynthetic superpowers?”**

The nanoceria are able to absorb infrared light, which no native plant pigments can do. Plant pigments like chlorophylls and carotenoids do a good job of covering the visible light spectrum, but this is only half of the incident solar energy beaming down on them all day. The researchers in this study conclude that the boost in photosynthetic capacity is in part due to the fact that the nanotubes absorb light energy beyond the visible range and can then transfer this energy to the photosynthetic machinery. In this way, the nanotubes are acting like an antenna- a giant antenna that doesn’t just boost the signals you’re already getting, but allows access to a whole set of new premium channels.

The nanotubes have a couple of other advantageous side effects as well. The cerium oxide nanoparticles are quite effective radical oxygen species (ROS) scavengers. Remember when I told you photosynthetic organisms have a complicated relationship with light? They need enough of it to live, but too much can be extremely damaging. This is why blasting plants with bright light at all times isn’t necessarily the best way of improving their growth and productivity. Likewise, improving their ability to capture light can also be dangerous. This is all due to the fact that overloading the photosynthetic circuitry with too many electrons can start to generate ROS which in turn irreversibly damage proteins, pigments and DNA they encounter. Since the nanoceria are quite good at scavenging or capturing and inactivating ROS, they also boost photosynthesis by helping out plants’ innate systems for dealing with this problem. Again, like Wolverine, plants have some level of ‘healing powers’ when it comes to dealing with light, but the nanotubes help kick it up a notch. Wolverine’s adamantium doesn’t really help with his healing powers, but coating his skeleton with it means it doesn’t get broken as easily and he doesn’t have to use his healing powers to mend broken bones. In the same way, the nanoceria prevent some damage before it happens so the plants don’t have to waste their resources on repairing damage caused by ROS.

In true superhero style, the nanotube-infused chloroplasts’ powers don’t stop there. They also confer the ability to detect certain chemicals in the environment. Yes, just like Wolverine has heightened senses (if he twitches is nose, he’s knows you’re there!), chloroplasts containing nanotubes do too. Different types of nanoparticles have been developed to detect toxins and pollutants like nitric oxide, sarin gas, and TNT. Integrating this technology into a photosynthetic organism may allow plants to become stealthy biodetectors of these chemicals. No longer would they be merely scenery, but solar-powered secret agents (green-ops?) with skills no training could ever provide.***

These super powers may not be indestructible retractable claws, but it’s a good start down the path to lots of useful applications.

Conclusion: Plants infused with nanoceria definitely qualify as super photosynthesizers, but we still have some work to do to put this into useful practice beyond the lab.

How do nanoparticles and enhanced photosynthesis affect plants over their entire lifespan vs. short experiments in the lab? Does this kind of boost in the light reactions translate into an increase in biomass? Sure, the light reactions could always use some help when sunlight is less than its brightest, but the real rate-limiting pathway is the dark reactions. (I’m looking at you Rubisco!).

How can this technology be translated into field agriculture to boost productivity of crops? Is it worth it? It’s not like you can propagate the nanotubes biologically, but who knows, maybe in the future tractors will be outfitted with attachments to infuse or spray seedlings with nanoparticles. In terms of calculating potential for improving plant productivity, photosynthetic energy conversion remains a variable in the equation that has yet to be thoroughly tapped when it comes to improving crop plants. Using carbon nanotubes to extend the spectrum of useful light into the infrared would definitely help plants breakthrough some yield ceilings we are seeing.

How can we turn our scenery into useful biosensors for pollutants? What kinds of chemicals can they detect and how can that be cheaply and easily measured? Sure, everyone and their grandma has a satellite and a drone these days, but are we really going to have to laser scan our environment every hour? How about a nice color change or wi-fi signal that NORAD can detect?

Speaking of wi-fi, why can’t we engineer plants with nanotechnology that allows them to have other superpowers, like transmitting wi-fi signals? Solar-powered free internet anywhere! Finally, getting plants to release something useful besides oxygen****, and it would go a long way to keep me from going over my monthly data plan limits.

These are all questions and exciting possibilities that will keep photosynthesis researchers like the Strano lab busy for years to come.

Johnna

*IMHO, ‘nanoceria’ is itself a name worthy of comic book lore. Maybe DC Comics can do a series where Swamp Thing is infused with nanoceria boosting his plant-like powers.

** You must be new here. Welcome.

***K-9 units may be relegated to history. SWAT, move over for SWNTs. (You’ll get it if you read the paper).

****It’s important to me that you catch the sarcasm in that phrase. If you’ve been breathing today, thank a photosynthetic organism.

References and Links:

http://phys.org/news/2014-03-bionic-synthetic-nanoparticles-photosynthetic.html

(paywall) http://www.nature.com/nmat/journal/vaop/ncurrent/full/nmat3890.html

http://www.popularmechanics.com/science/health/genetics/the-worlds-first-bionic-plant-16604565?click=pm_latest

http://www.scientificamerican.com/article/bionic-plants-offer-superpowered-photosynthesis/

 

Some Answers for Q

Today’s post features new work from Liu et al published in PNAS (paywall). Since the subject is near and dear to my scientific heart, it’s also a mash-up of some of my work too. My favorite enzyme Photosystem II (PSII) uses sunlight to split water to make oxygen and shuttle electrons down the photosynthetic electron transfer chain. So, yeah, it’s kinda important and lots of scientists (really, not just me) would like to know everything about how it functions.

Knowing the structure of this large complex helps our understanding of how it works. PSII has an extensive parts list (more than 20 proteins and even more cofactors like pigments and metals). The proteins are named PsbA-PsbZ, Psb27 and the numbers increase numerically as we continue to identify subunits. It’s quite the alphabet soup. This post is brought to you by the letter Q- PsbQ, that is. I’ll bring you up to speed on what we know about this protein and what new answers we have from Liu et al’s new publication.

PsbQ is a small protein associated with the lumenal side of PSII. There are quite a few differences in extrinsic protein composition in plants vs. cyanobacteria. Researchers have long known about the PsbQ protein in plants, but a similar protein has only more recently been identified in cyanobacteria.

Differences in the lumenal extrinsic proteins, plants vs. cyanobacteria

Differences in the lumenal extrinsic proteins, plants vs. cyanobacteria

Some of my thesis work involved characterizing the specific defects of cyanobacterial PSII in the absence of the PsbQ protein. When PsbQ isn’t around, the effects are subtle but significant. The PSII complex isn’t quite as stable and has lower activity under stress conditions like nutrient limitations. To truly peg the PsbQ protein as a legitimate PSII subunit and not just some co-purifying protein with modest effects on PSII function, I resorted to biochemistry (of course!). The common way of purifying PSII with a his-tagged CP47 (PsbB) protein results in PsbQ as a co-purifying protein. I created a his-tagged version of PsbQ and purified PSII. This reciprocal purification experiment demonstrated that PsbQ was indeed a PSII subunit. Not only that, but the PSII complexes I purified were much more stable and had higher activity than the CP47-tagged complexes. However, I recovered much less PsbQ-tagged PSII relative to my purifications of CP47-tagged PSII. These results were interpreted to mean that the PsbQ-containing PSII complexes were the subset of PSII in a cell that were fully-assembled and functional. On the other hand, CP47-containing PSII complexes represent a broader spectrum of PSII complexes in varying states of assembly; as such, their overall activity and stability is lower than the PsbQ-containing PSII. My work also showed some effects of the loss of PsbQ on the PsbV protein, so I made a cartoon model for PSII assembly that looked like this:

The PSII subunits are colored as follows, D1 (light green); D2, Cyt b559, CP43 (dark green); CP47 (cyan); Psb27 (pink); PsbO (teal); PsbV (dark blue); PsbU (light blue); PsbQ (purple); and damaged D1 (yellow).  The top half of the cycle represents steps in the synthesis half of the pathway resulting in fully assembled dimers on the far right, and the bottom half of the cycle shows the disassembly of the complex and removal of the damaged D1 protein.

The PSII subunits are colored as follows, D1 (light green); D2, Cyt b559, CP43 (dark green); CP47 (cyan); Psb27 (pink); PsbO (teal); PsbV (dark blue); PsbU (light blue); PsbQ (purple); and damaged D1 (yellow). The top half of the cycle represents steps in the synthesis half of the pathway resulting in fully assembled dimers on the far right, and the bottom half of the cycle shows the disassembly of the complex and removal of the damaged D1 protein.

So this is what I thought the PsbQ-containing complexes looked like:

PsbQwrong

Despite the experimental evidence that PsbQ is a legitimate PSII subunit, it is not present in our current structural models. For a number of technical reasons, the PsbQ protein just doesn’t exist in the PSII material that forms crystals that are used to make our structural models. This is a problem for researchers like me because I’d like to know where PsbQ is relative to the rest of PSII. Liu and co-authors* addressed this question using a different experimental technique. They treated PsbQ-containing PSII complexes with chemicals that would covalently crosslink protein components with one another. Then they used mass-spectrometry to identify which protein fragments got linked to one another and where.** Once a handful of crosslinks were identified (PsbQ+PsbO, PsbQ+CP47, even PsbQ+PsbQ***), these data create constraints on the position and orientation of the PsbQ protein.

How is this possible? Other researchers have experimentally determined the structure of the PsbQ protein by itself (not part of the PSII complex). I’ve mentioned before that we know the structure of cyanobacterial PSII without PsbQ.

These structures are combined with one another and modeled to take into account the constraints of the cross linking data to show where PsbQ resides within PSII. So, where is it? Sitting right on top of the PsbO protein near the interface of the PSII dimer. (yes, there’s a much better figure in the paper.) How does that compare to my cartoon model from 2007? Way off. (See above)

PsbQright

So what new answers do we have for PsbQ?

Evidence suggests that the PsbQ protein interacts closely with the PsbO and CP47 proteins (and even with another copy of itself) at the PSII dimer interface. Liu et al did some additional biochemical work to sort out the relationship between PsbO and PsbQ. As it turns out, the PsbO protein is required for PsbQ association.

This isn’t the end of the story for PsbQ. In science, the quest continues. Here are my new Q’s for PsbQ:

What about the relationship between PsbQ and PsbV? There is no evidence from cross-linking that these proteins physically interact, but other biochemical analyses suggest that the absence of PsbQ affects the stability of PsbV. Is there a physical association that could not be detected using this crosslinking method (i.e. no reactive residues on both proteins within the right distance or other technical issues with detection)? Alternatively, are there widespread conformational changes within PSII when PsbQ binds that also effect PsbV?

What’s really going on with the N-terminus of PsbQ? In the structure and the latest model, it just floats off from the helices into oblivion. It’s unlikely that this represents the physiological state of the N-terminus of PsbQ in vivo. The structure probably tightens up once it makes contact with other components of PSII. Also, the N-terminus of the mature protein has a lipid modification that anchors it within the membrane. The model of PSII with PsbQ probably still has some wiggle room in it to stretch the N-terminus so that the end reaches the thylakoid lipid bilayer.

What does this mean for higher plants? Is PsbQ in an analogous position close to PsbO in plants? These proteins are common between plants and cyanos, but their sequences are quite different. Could the overall structure be conserved even though the specifics are not?

We may have some new answers, but there are always more questions to pursue.

Johnna

*This is work Haijun did while in the Pakrasi Lab (my thesis lab) at WashU. It’s a good thing my lab notebooks so clearly described everything I had done and where my strains were in the freezer!

**If this is starting to sound like déjà vu, you probably read my post Photosynthesis Goes Voltron. It was also work by Haijun Liu using chemical crosslinking and mass spec techniques to identify interactions among other protein complexes. It truly is a powerful technique with lots of applications especially in the realm of difficult-to-work-with protein complexes. Also, this makes him the first author to be highlighted twice on my blog. I’m sure he’s updating that under the ‘awards’ section of his CV.

***Is that legal? Yes, proteins can crosslink to themselves if two reactive residues are close enough to one another. In this case, they are nowhere near one another and can only be explained by an interaction between two separate PsbQ molecules, which must be in close contact with one another.

References and Links:

http://www.pnas.org/content/early/2014/02/13/1323063111.abstract

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/

Arbor Day: Thinking about the Trees for the Forest

treeThis weekend in Louisiana, we are celebrating Arbor Day. Some of you reading this from more northerly latitudes may think it’s absurd to try to dig a hole in the ground in January and a fool’s errand to get anything would take root and live in it. Nevertheless, late winter/early spring is the perfect time for tree planting in Louisiana because it gives the roots several months to establish growth in the new location before our oppressive summer heat sets in. Check out this link for species recommendations and planting tips. Also, for those of you in the Baton Rouge area, the LSU AgCenter Botanic Gardens at the Burden Center will be having a full day of arboreal festivities. If your family plants a tree in the Burden Woods, you get GPS coordinates so you can keep track of your tree’s growth over the years.

If you live in a place where you are still regularly using an ice scraper on your car, don’t plant a tree today. Please consult this link to determine your Arbor Day date. You still have time to order tree saplings from the Arbor Day Foundation that will be best suited for your growing area. For example, if this weekend is not your Arbor Day, don’t order lemon or other citrus trees. Also, if you are inclined to become a Foundation member, you can even get free trees. Keep an eye out for Arbor Day events at your local nurseries, agricultural extension offices and botanical gardens.

I know that Arbor Day is all about planting new trees, but new research published in Nature this week* should change the way we think about old trees too. Yes, get ready for something new about something old under the sun. In case you’ve never thought about it, plants are very different from us when it comes to growth and development. Many plants and trees in particular just keep growing and getting bigger. Researchers had known that trees reach an ‘adolescent growth spurt’ when they grow faster after they’ve reached a certain size, but it had always been assumed that as they get much older their rate of growth slows down. Scientists surveyed more than 600,000 older tree specimens across the globe of more than 400 different species to check this assumption. Crunching all of these numbers on growth rates yielded a surprising conclusion- the majority of older trees were accumulating mass at an increasing rate. In other words, there’s no slowing down in the golden years for these trees.

On a forest scale, younger forests are better (compared to older forests) at trapping carbon dioxide from the atmosphere because there are more trees in a given area for these forests. This new research means reconsidering how individual trees are contributing to this process. On a per tree basis, the data says that older trees are pulling in much more carbon dioxide and converting that to tree mass. Of course, when these large, older trees fall**, the potential for carbon dioxide release is also greater because more is produced as a byproduct of the decomposition process. This is likely why the fast growth of older trees was masked in forest-scale measurements of primary productivity comparing simply ‘young’ vs. ‘old’ forests. These new findings will change the way we think about forest management. When thinking about maintaining optimal productivity of forests, it’s important to keep these older trees instead of culling them.

So, today or whenever your Arbor Day is, make sure you celebrate by planting a new tree and appreciating the older trees in your environment. For more inspiration on superlative trees today, check out my ‘Super Photosynthesizer‘ posts on Hyperion (The World’s Tallest Tree) and Methuselah and Prometheus (World’s Oldest Living Organisms).

Johnna

*Unfortunately, the original research paper is behind a paywall, but check out some of the other links below for more freely-available commentary.

**Whether or not anyone hears it.

References and Links:

http://www.lsuagcenter.com/news_archive/2013/december/headline_news/Arbor-Day-celebration-set-for-Jan-18-at-Botanic-Gardens-.htm

http://www.lsuagcenter.com/en/our_offices/research_stations/Burden/News/Arbor-Day-2014.htm

http://theadvocate.com/features/people/7949344-123/br-getting-ready-for-arbor

http://www.nature.com/news/tree-growth-never-slows-1.14536

http://www.nature.com/nature/journal/vaop/ncurrent/full/nature12914.html

http://www.npr.org/2014/01/16/262479807/old-trees-grow-faster-with-every-year

http://www.scientificamerican.com/article.cfm?id=older-trees-grow-faster-as-they-age

http://www.livescience.com/42578-oldest-trees-grow-fastest.html?cmpid=514645

http://www.usgs.gov/newsroom/article.asp?ID=3781

Even microbial marriages are more than they seem

I wrote a guest post for the American Society of Microbiology blog Small Things Considered. Click the link to read more about the intimate relationship between a hardworking photosynthesizer and its motile heterotrophic partner. What do these two species see in one another? Has a metabolically lazy flagellate found biochemical sugar daddies? Have green sulfur bacteria engineered a way to steer a motile bacterium to their favorite places? Don’t be so quick to judge… the answers will surprise you.

How do they make it work? Genomic Revelations on a Microbial Consortium in Small Things Considered

Johnna

Original journal article: http://genomebiology.com/2013/14/11/R127