Category Archives: inspiration

Pyramid of Biochemistry Greatness

As I mentioned in my previous post, there’s at least one person in the world searching the internet with the phrase ‘everything aspiring biochemists should know.’ I feel obligated to share my pyramid for biochemist success. It’s based on Ron Swanson’s perfectly calibrated recipe for personal achievement. Click this link for context if you are not familiar with Parks and Rec.

Pyramid

Here is a link to the PDF with a little better resolution. Now, go achieve your dreams. I would say good luck, but as Ron says, “Luck is a concept created by the weak to explain their failures.”

Johnna

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

The Grinch Who Stole Science

The Grinch may be green, but he’s not photosynthetic.* However, here’s a grinchy science parody that fits just perfectly on this blog.

 

Every Sci down in Sci-ville liked Science a lot… But The Grinch, who lived just North of Sci-ville, Did NOT! The Grinch hated Science! The whole research season! Now, please don’t ask why. He just wouldn’t listen to reason. It could be that his head wasn’t screwed on quite right. It could be, perhaps, that his ties were too tight. But I think that the most likely reason of all may have been that his brain was two sizes too small. But, whatever the reason, his brain or his ties, he stood there on the funding committee, hating the Scis.

Staring down from his office with a sour, Grinchy glower at the warm lighted windows below in their tower. For he knew every Sci was preparing their aims, busily now supporting their claims. “And they’re gathering their data!” he snarled with a sneer. “Deadline’s tomorrow! It’s practically here!” Then he growled, with his grinch fingers nervously drumming, “I MUST find a way to keep Science from coming!” For, tomorrow, he knew… …All the Sci girls and boys would wake up bright and early. They’d rush for their grants! And then! Oh, the experiments! Oh, the experiments! Experiments! That’s one thing he hated! The Experiments! Then the Scis, young and old, would sit down to a bench. And they’d research! And they’d research! And they’d research! Research! Research! They would start on the -omics, and screens on yeast which was something the Grinch couldn’t stand in the least!

And THEN They’d do something he liked least of all! Every Sci in the tower, the tall and the small, would stand close together, with hypotheses greeting. They’d stand hand-in-hand. And the Scis would start meeting! They’d meet! And they’d meet! AND they’d meet! Meet! Meet! Meet! And the more the Grinch thought of the Sci’s Annual Meeting, The more the Grinch thought, “I must stop this whole thing! “Why for fifty-three years I’ve put up with it now! I MUST stop Science from coming! …But HOW?” Then he got an idea! An awful idea! THE GRINCH GOT A WONDERFUL, AWFUL IDEA!

“I know just what to do!” The Grinch laughed in his throat. And he made a quick Scientist lab coat. And he chuckled, and clucked, “What a great Grinchy ploy! “With this coat, I’ll look just like a PhD decoy!” “All I need is a postdoc…” The Grinch looked around. Since postdocs are cheap, one could easily be found. But they wanted benefits? No! The Grinch simply said, “If I have to pay a postdoc, I’ll make one instead!” So he called his dog Max. Then he took some red thread and he tied a big hood on top of his head. THEN He loaded some bags and some old empty sacks on a research vessel and he hitched up old Max. Then the Grinch said, “Giddyap!” And the vessel started down toward Sci-ville where the Scis lay a-snooze in their labs.

All their windows were dark. Quiet snow filled the air. All the Scis were all dreaming sweet dreams without care when he came to the first lab in the square. “This is stop number one,” The old Dr. Grinchy hissed and he climbed to the roof, empty bags in his fist. Then he slid down the fume hood, a rather tight pinch, but if phenol could do it, then so could the Grinch. He got stuck only once, for a moment or two. Then he stuck his head out of the fume hood flue where the little Sci flasks all hung in a row. “These flasks,” he grinned, “are the first things to go!” Then he slithered and slunk, with a smile most not nice, around the whole room, and he took every device! Beakers! And pipettors! Stir plates! Tris! Sequencers! Manuscripts! Timers! UV-Vis! And he stuffed them in bags. Then the Grinch, very nimbly, stuffed all the bags, one by one, up the fume hood! Then he slunk to the freezer. He took the Scis’ box! He took all the strains! He took the lab stocks! He cleaned out their chem shelf as quick as can be. Why, that Grinch even took their last can of LB!

Then he stuffed all the data up the fume hood with a rant. “And NOW!” grinned the Grinch, “I will stuff up the grant!” And the Grinch grabbed the grant, and he started to shove when he heard a small sound like donning a glove. He turned around fast, and he saw a small Sci! Little Cindy-Vi Sci, who’s qual exams were nigh. The Grinch had been caught by this little Sci daughter who’d got out of lab for a cup of DI-water. She stared at the Grinch and said, “Reviewer, why, “Why are you taking our R01 grant? WHY?” But, you know, that old Grinch was so smart and so slick He thought up a lie, and he thought it up quick! “Why, my sweet little tot,” the fake PhD lied, “There’s a fatal flaw in this aim that’ll kill the whole grant. “So I’m taking it home to triage, my dear. “I’ll fix it up there. Then I’ll bring it back here.” And his fib fooled the child. Then he patted her skull and he sent her to rethink of her hypothesis null. And when Cindy-Vi Sci went away with her thoughts, HE killed the grant until their funding was naught! Then the last thing he took was the log for their data. Then he went up the fume hood himself, the old hater.

On their walls he left nothing but hooks, and some wire. And the one speck of media he left in the lab was a crumb that was even too small for a stab. Then He did the same thing to the other Scis’ labs leaving crumbs much too small for the other Scis’ stabs! It was quarter past dawn… All the Scis, still a-bed all the Scis, still a-snooze when he packed up his vessel, packed it up with their instruments! Reagents! The tweezers! The tape! And the shakers! The centrifuges! The freezers! Three thousand feet up! Up the side of Mount Rejectit, He rode to the tiptop to eject it! “Bye-bye to the Scis!” he was grinch-ish-ly humming. “They’re finding out now that no research is coming! “They’re just waking up! I know just what they’ll try! “Their mouths will hang open a minute and sigh “Then all the Scis down in Sci-ville will all cry Why-why!”

“That’s a noise,” grinned the Grinch, “That I simply must hear!” So he paused. And the Grinch put a hand to his ear. And he did hear a kick-starter rising over the snow. It started in low. Then it started to grow… But the sound wasn’t sad! Why, this sound sounded rational! It couldn’t be so! But it WAS full of hope! No dope! He stared down at Sci-ville! The Grinch popped his eyes! Then he shook! What he saw was a shocking surprise! Every Sci down in Sci-ville, the tall and the small, was researching! Without any grants at all! He HADN’T stopped Science from coming! IT CAME! Somehow or other, it came just the same! And the Grinch, with his grinch-feet ice-cold in the snow, stood puzzling and puzzling: “How could it be so? It came without R01s! It came without panels! “It came without fellowships or reviews through normal channels!” And he puzzled three hours, `till his puzzler went can’t. Then the Grinch thought of something he hadn’t before! “Maybe Science,” he thought, “doesn’t come from a grant.”

Maybe Science…perhaps…means a little bit more!” And what happened then…? Well…in Sci-ville they say that the Grinch’s small brain grew three sizes that day! And the minute his brain didn’t feel quite so tight, He whizzed with his load through the bright morning light and he brought back the equipment! And the laserjet toner! And he… …HE HIMSELF…! The Grinch became a top donor!

 

Johnna

*I just wanted to clearly state that since I know at least one person stumbled upon my blog with the search term “Is the Grinch Photosynthetic?”

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

Behind the Music: Plants

I’ve spent a lot of time on this blog writing about the importance of plants. They are the energetic foundation of our biosphere. We use them for food, fiber, fuel and building materials. This is why it is so important to understand how they work to further extract useful work and energy out of them. Today’s post explores an area that plants have not been adequately exploited, and I have no idea why we haven’t jumped on this sooner. I’m talking of course about music production.

Before you can even utter LOLWut!?!, allow me to introduce Data Garden. They are a group dedicated to creating new electronic art and have successfully completed a Kickstarter campaign to fund MIDI Sprout. It’s a cool idea with an even cooler logo (also true for Data Garden generally). MIDI Sprout is a device that allows users to create biofeedback art from plants. Huh? You attach the electrodes to the leaves of your houseplant and it records the changes in electrical signals emitted by the plant into an output readable by synthesizers and computers. So, basically it allows you to turn your plant’s existence into music. They’ve already had an exhibit at the Philadelphia Museum of Art and the recording is available for purchase on their website.

So many thoughts on this…

This may blow your mind, but so many of us plant scientists, photosynthesis researchers specifically, have been communicating with our plants using light and fluorescence to determine how healthy or sick they are. We haven’t been listening to them. Maybe I can finally get a Science or Nature paper if my methods include synthesized acoustics as an assay for plant fitness. Those journals just fall all over themselves when it comes to new methods. The evil scientist in me wants to hook up my lab plants to the MIDI Sprout electrodes and run all of my usual experiments and treatments- red light excitation, treatment with the herbicide DCMU, plant hormones, methyl viologen, DBMIB, or high or low CO2 conditions. I would also require an instrument with submersible electrodes so I can record my algal and cyanobacteria cultures as well. Why should plants be the only photosynthetic organisms to hit the top 100?

The MIDI Sprout is advertised as an instrument to listen to your houseplants, but I don’t think my singular Cristmas cactus will cut it. I’m sure it’s just because you need a pluggable power source, but a portable version can probably be developed with enough solid state storage capacity to cache recorded responses until you can get back to the studio and remix everything. Then you can listen to gardens, natural environments, GMO cornfields or whatever you want. Move over Sound Garden, here comes Garden Sound. Interestingly, there are already quite a few musical recordings close to the genre if you search iTunes for ‘photosynthesis.’ They Might Be Giants has a catchy song about photosynthesis on their ‘Here Comes Science’ album. There’s also a group called Carbon Based Life Forms experimenting in the new age genre with plant-related themes. There’s even a group called ‘Plants’ on the new age label Strange Attractors in the same spirit. However, these are all humans as far as I can tell- autotroph imposters or interpreters- and not actual plants.

Plant-based music could introduce entire new genres and bands. What could they be called? Well ‘photosynthesizers’ is a little obvious and hack and I use it for something else. Phonosynthesis is already taken (recommend the album BTW). I’m copyrighting the term ‘autotrophony’ today as this new music genre. I’m sure it will stick if I hashtag it up on the interwebz. Regardless just think of the possibilities… Chard in G minor, Cacti concerto, Solanum sonata, tulip tunes, floral phonics. Autotrophic American Idol. Move over Beyoncé, here comes Botanée. Producers could create the perfect plant boy band equivalent with different potted species with no chance of a break-up. Can’t storm off stage if you’re immobile! If you hated Monsanto as an agribusiness empire, just wait until they break into the music industry. They will surely negotiate for royalties on music made by plants their seeds produced.

I’m thinking we could kick this movement into the mainstream. Some of the plant-synthesized music has some potential. It just needs some help from our species, since we will be the ones purchasing it. Collaborations with existing artists are what we need. Just think- there could be actual black-eyed peas on The Black Eyed Peas album. Remixes are where it’s at, so I just need the Skrillex Ilex, Avicii Vitis or David Guetta Betula remix. I’m thinking clap tracks ala LMFAO and vocals by Pitbull and Ke$ha. There’s gotta be a hit in there somewhere. I really just need to be a one-hit wonder to fund my photosynthesis research for the rest of my career, but short of that I could probably just DJ weddings, parties and bar mitzvahs on the weekends to independently sustain my lab outside of federal funding dollars.

Dr. Z Scheme PhD Sigh, there were no female DJ clip art images.

Dr. Z Scheme PhD
Sigh, there were no female DJ clip art images.

Of course, there are other artistic experimental ventures I could do with this system. I could try something totally meta. Remember that Talk to a Plant museum exhibit aimed at influencing plant growth with sound? What if you played music to plants while you were recording them for their music? Would it sound similar to the music in the room? Would those recordings be different than the music plants make in silence? Yeah. Mind. Blown.

Why does it have to be just an auditory experience? Why can’t you record the music of your food plants then eat them while listening to their music. I’m also trademarking the ‘Salad Soundtrack Bistro.’ If you live in a state like Colorado, recordings could be made of certain plants used for other recreational purposes and they could be sold as a packaged altered experience. Note, it’s just a good business plan to put my Music Munchie Bodega next door.

Clearly, I have tons of creative ideas for future plant exploitation for the sake of the arts and making money. I’m very curious as to the general availability of the MIDI Sprout on the horizon. If any readers have connections in the music industry, tell them to contact me. Otherwise, I guess I will have to figure out how to start my own youtube channel to get noticed. My music mogul persona is Dr. Z-scheme PhD on my P680 Fluorescent label.* Let’s do this.

 

Johnna

*My plant science nerd friends reading this will get it.

References and Links:

http://www.theguardian.com/technology/2014/may/08/cosmic-soundscapes-plants-midi-sprouts

http://www.dazeddigital.com/artsandculture/article/19191/1/midi-sprout-uses-houseplants-to-play-synth-electronic-music

https://www.kickstarter.com/projects/datagarden/midi-sprout-biodata-sonification-device

http://datagarden.org/7940/midisprout/

http://www.youtube.com/watch?v=WN833bCXXJk

http://datagarden.org/about/

Spring-spiration: Plant Color

I’m hosting April’s Berry Go Round blog carnival and the theme is plant color.* So writers of plant science, nature and gardening: whip up something entertaining, informative and colorful (new or old) and submit your links here by the 26th of the month. You can talk about your favorite colors, unusual colors, pigment biosynthesis, how plants use color, how humans have painted new colors onto our favorite plants, color patterns, temporal color changes etc. The canvas is blank and waiting for you. Of course, I’m supportive of posts about all photoautotrophs for those adventurous writers daring to step away from the plant kingdom and delve into algae, cyanobacteria and other microscopic photoautotrophs. Heck, if your post is about fungi, you’ll probably get a pass if it’s colorful enough. I will compile them all together in a link-fest blog carnival by the end of the month.

Spring is definitely here to stay at my latitude, so here are some colorful pictures for inspiration.**

azalea Digitalis Johnny Jump Ups Digitalis Rhododendron bearded iris wisteria iris red clover

 

Johnna

*The March Berry Go Round featured posts about Unusual Edibles. Check it out: http://emmacooper.org/blog/march-berry-go-round-unusual-edible-plants

**All photos taken by myself at Afton Villa Gardens in St. Francisville, LA

The Twelve Days of Christmas Plants: Chestnuts

This series of posts will highlight the plants that help you celebrate the Yuletide season.

“Chestnuts roasting on an open fire

Jack Frost nipping at your nose

Yuletide carols being sung by a choir

And folks dressed up like Eskimos”

The Christmas Song


American Chestnut Nuts with Burrs and Leaves. ...

American Chestnut Nuts with Burrs and Leaves. Photo by myself: Timothy Van Vliet 2004 from my Orchard in New Jersey (Photo credit: Wikipedia)

The lyrics to The Christmas Song were written during the summer of 1944 and recorded by Nat King Cole in 1946. They were meant to be the acme of the spirit of the winter season. How much do you know about the tree behind the tradition? The American Chestnut’s story of loss and the brink of hope seems almost too fitting for the Christmas season.

By the time those lyrics were written, the American Chestnut (Castanea dentata) was nearing extinction. The forests of the Eastern United States were once filled with this fast-growing species with numerous specimens reaching nearly 100 feet tall and 10 feet in diameter. It provided sustenance for deer, turkeys, bears and passenger pigeons. Its wood was used for a variety of purposes since it was both lightweight and strong. Logging was not the cause of the demise of the American Chestnut.

American Chestnut, Central Maryland. Photograp...

American Chestnut, Central Maryland. Photograph supplied by the United States Forest Service. (Photo credit: Wikipedia)

English: Range map of American chestnut (Casta...

English: Range map of American chestnut (Castanea dentata). (Photo credit: Wikipedia)

In 1904, American Chestnuts in the Bronx Zoological Park were reportedly dying, infected with a Chetsnut blight fungus (Cryphonectria parasitica). It was determined that this pathogen had been imported to the United States along with chestnut trees from Asia. Because those varieties had long coexisted with the fungus, they had immunity to the disease, but our American Chestnuts were woefully susceptible. By the 1940s, the fungus had spread throughout the entire range of the American Chestnut. Once infected, the trees died within a decade as the fungus cuts off the lifeline connecting the photosynthetic leaves to the rest of the tree. Because the fungus didn’t completely kill all the roots, the trees vainly sprouted new shoots from the stumps, but eventually these too would fall victim to Chestnut blight. By 1950, nearly 4 billion trees were dead. The loss of the American Chestnut not only changed a Christmas tradition, but also drastically altered the composition of our Eastern forests as slower-growing species like oaks began to fill the empty ecological niche.

Very few specimens of American Chestnuts survive today, and their discovery is always newsworthy. Adult trees have been identified in Ohio, Tennessee, Alabama, Kentucky, Pennsylvania and Georgia. These specimens have escaped infection for a variety of reasons including micro-niche conditions and possibly superior genetics. The largest stand of American Chestnuts can be found in West Salem, Wisconsin. These were planted by an early settler in the area, and because they are outside the natural range of the American Chestnut, they largely escaped the blight. However, in 1987 scientists discovered that even these trees had become infected, and they have been the focus of intense research efforts to save them.

A number of different strategies are being used to ensure that the American Chestnut does not permanently disappear. The American Chestnut Foundation is coordinating restoration efforts. One strategy involves back-crossing American Chestnuts with the blight-resistant Chinese Chestnut. By crossing these two varieties, disease-resistance is conferred. These offspring are then back-crossed to the American Chestnut six more times, selecting for blight-resistance along the way. By 2005 the program had yielded the first blight-resistant chestnuts that were 15/16ths American Chestnut. Because these offspring are 94% American Chestnut, they retain many of the properties of this species. These are being re-introduced in test plots by the U.S. Forest Service. As of this year, nearly 80% of the back-crossed saplings are surviving. Other breeding studies are being conducted with the remaining wild specimens of American Chestnut trees.

chestnut

More modern technologies are even being applied for the salvation of the American Chestnut. In an NSF-funded project, the genomes of various tree species including the American and Chinese Chestnuts have been sequenced in order to identify the specific genes involved in blight-resistance. This knowledge will provide better genetic markers for screening in traditional breeding programs as well as target genes for engineering. To facilitate these efforts, researchers have developed new techniques for propagating and transforming American Chestnut plant material. As scientists learn more about plant disease resistance in other plant species (like crop plants), those genes also become candidates for engineering into the American Chestnut. For example, transgenic American Chestnuts containing a gene from wheat that helps confer resistance have been planted for field tests. Hopefully the genome of the Chinese Chestnut will reveal additional resistance genes that can be introduced transgenically.

Scientists are also using other methods to tame the Chestnut blight fungus. One way to combat the microbe that felled the giant trees is by using another microbe- a naturally-occurring virus that attacks the Chestnut blight fungus. This is a method that may save mature trees already infected with the fungus like those in West Salem, Wisconsin. It works to heal infected trees in a way similar to modern human vaccinations. Scientists isolate the virus from other infected American Chestnuts and inoculate blight-infected trees. The virus weakens the fungus enough so that the trees can recover. The initial results have proven promising, but this is not the magic bullet solution that they were hoping for.

With all of these efforts, roasting chestnuts over an open fire may once again be a common American Christmas tradition, but science still has a long way to go to bring this species back from functional extinction. Even then, it remains to be seen whether crossbred or transgenic (aka GMO) American Chestnuts will be accepted by the general public. However, there does seem to be some poetic justice in a restoration solution that involves a small piece of foreign DNA to protect these giant trees from a foreign deadly microbe.

Johnna

References and Links:

http://dendro.cnre.vt.edu/forsite/paul/paul1.htm

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

http://www.esf.edu/chestnut/background.htm

http://www.fs.fed.us/r8/chestnut/qa.php

http://dnr.wi.gov/wnrmag/html/stories/2002/aug02/chest.htm

http://aobblog.com/2013/10/american-chestnut-returns/

http://www.acf.org/pdfs/education/TheLegendoftheAmericanChestnutTree.pdf

http://www.acf.org/pdfs/about/restoration.pdf

http://www.acf.org/pdfs/news_room/Blight%20Resistance.pdf

http://blogs.usda.gov/2013/12/06/restoration-efforts-may-mean-more-chestnuts-roasting/

http://phenomena.nationalgeographic.com/2013/03/11/resurrecting-a-forest/

http://www.esf.edu/chestnut/genes.htm

http://www.esf.edu/chestnut/tissue-culture.htm

http://www.fagaceae.org/outreach

http://www.nature.com/news/plant-science-the-chestnut-resurrection-1.11504