Root to Shoot Calcium Wave

While a major research effort in the Gilroy Lab involves studying how plants grow in microgravity on the ISS, equally huge is ongoing plant research on the ground here in our lab at UW-Madison.

Last week, Won-Gyu’s paper on calcium signaling in Arabidopsis was published in the Proceedings of the National Academy of Science (PNAS). He discovered that when roots are hit locally with salt stress, a calcium wave quickly spreads from the point of stress throughout the whole plant. This calcium wave plays a role in how plants communicate within themselves to coordinate a response.

UW-Madison communications posted a write-up about our calcium research today. Feel free to ask a question or to leave a comment below. Enjoy!

tpc

Arabidopsis thaliana growing in salt water. The plant on the right has more of the protein channel TPC1, thought to be involved in calcium signaling in plants. The plant at center has less TPC1 than normal, and the one on the left is considered normal.

 

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BRIC-17 Data Analysis

In the past few months, the Gilroy Lab space biology research team has been busy analyzing data from our space-grown and Earth-grown control plants from our BRIC-17 TOAST experiment. Excitingly, we now have a full set of RNAseq data! The RNAseq is important to our analysis because it will give us insight into the differences in gene expression of plants grown in microgravity compared to ones grown on Earth and allow us to ask our big question: “Do plants grown on the ISS look like plants experiencing low oxygen stress on Earth”?

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The International Space Station seen as it is orbiting Earth.

We presented some of our preliminary results at the American Society of Gravitational and Space Research  meeting in Orlando at the end of last year. This meeting is the primary venue for space biology researchers to report their results to the rest of the space research community, a community that includes NASA-funded researchers who work for universities or for private companies. This was a fantastic meeting that included not only physicists and biologists who conduct research in microgravity but also NASA employees who run the research programs and engineers who develop hardware for research on the ISS. Hanging out with rocket scientists is an incredible amount of fun!

American Society for Space and Gravitational Research

American Society for Space and Gravitational Research

As a reminder, here are the primary questions we wanted to investigate for our BRIC-17 TOAST experiment growing plants on the space station. For more details about rationale and preliminary data leading to this experiment, see the “Experiment” details page.

  • Do hypoxic (low oxygen) conditions develop in plants grown in microgravity aboard the ISS?
  • Does cax2, a plant lacking a protein which transports calcium, allow for improved plant growth under these conditions?

First, we wanted to quantify any size differences between the plants grown in microgravity compared to ones grown on Earth. For this, Won-Gyu lined up the seedlings from each petri plate and took digital images. With help from an image analysis program, he measured the total size of each seedling and took individual size measurements from each shoot and each root.

measurement method

Measuring root and shoot lengths of flight-grown material.

It turns out that the cax2 mutant seedlings from the Space Station do show different sizes of roots and shoots, so it looks like at least some of our ideas about low oxygen stresses in space may be correct.

Second, Won-Gyu looked at changes in the expression of specific genes, genes which we know have altered expression levels in a plant experiencing low oxygen stress thanks to other Earth-based plant research. He did this analysis by isolating RNA from our seedlings and using quantitative PCR (qPCR) to detect how much RNA was present. Looking at the amount of transcribed message (mRNA) from these specific genes will give us insight into the magnitude of hypoxia experienced by plants grown in microgravity compared to Earth-grown seedlings. Again, it turns out that the level of some, but not all, genes related to low oxygen stress on Earth are altered in spaceflight and are different in the cax2 mutants from the Station. We are now in the the most exciting aspect of research; it looks like some of our predictions are correct but the plants are telling us something extra that we need to try and understand.

These qPCR experiments investigated a few select genes already characterized to be involved in the plant hypoxic response.  However, the RNAseq data allows us to explore how spaceflight affected the levels of all 27,000 genes present in Arabidopsis. This RNAseq analysis is going to be the way we will tease apart what was going on to these plants aboard the ISS. As you can imagine, this set of data is huge (for the computer geeks out there, we have about 1 terabyte of raw data to sift through). Analyzing these results is in progress, but we hope to finish the work by the time spring arrives in Madison!

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Spacefaring Vegetables

If you are in Madison, Simon will be giving a presentation at the UW Space Place Dec 10 at 7pm. “Spacefaring Vegetables: Why Does NASA Launch so Much Lettuce?”
http://www.spaceplace.wisc.edu/#guest

Plants and microbes keep us alive on Earth; they feed us and they purify our air and water. Could they do the same for a long duration spaceflight or a colony on Mars? But how does a lack of gravity in space affect plants, microbes and humans? Prof. Gilroy will try answer what happens to biology when it is in spaceflight and whether down always needs to be down and 1x gravity is always good.

I’m told that it will be recorded by Wisconsin Public Television and posted online. I’ll let you know when the link to the video becomes available.

Image

The Space Place is located at 2300 S. Park Street in the Villager Mall, just north of the Beltline Highway.

Categories: Plants in Microgravity | 2 Comments

ASGSR Webcasts

The annual meeting of the American Society of Gravitational and Space Research (ASGSR) is coming up next month, November 4th through 8th. This year, the meeting will be held in Orlando, Florida. A vast number of scientists involved in NASA-funded research (space station experiments in addition to ground-based projects) will present their most current results at this meeting.

The three of us from the Gilroy Lab involved in the BRIC-17 experiment will be attending the ASGSR meeting. In particular, Simon will be organizing a special session: “Introduction to Physical and Life Science Microgravity Research.” Because the society recently expanded to include scientists interested in both biology and physics, there was a need to present the key problems and questions of both areas in a compact way understandable by both groups of researchers. Simon will be teaming up with physicist Mike Banish from the University of Alabama at Huntsville to give the talk at this special session.

This session may also be of interest to many folks following this blog who are not attending the meeting because it will be a more general overview of microgravity research. Luckily, this session will be part of the ASGSR meeting webcast, so you too could “attend” the talk which is scheduled for 8am eastern time on Thursday November 7th.

For information about how to access the webcast, you should check the ASGSR website.

American Society for Space and Gravitational Research

American Society for Gravitational and Space Research

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BRIC-17 Deintegration

The Dragon capsule’s release from the space station in March was delayed by a day due to rough seas in the area planned for the splashdown, otherwise the release and splashdown went exactly as planned. If you are curious about seeing how Dragon leaves the ISS, here is a time-lapse video showing the process. It is pretty amazing when you remember this is all going on at 5 miles per second, 250 miles above our heads.

Following splashdown in the Pacific, the Dragon capsule was loaded onto a ship and transported to California. While en route, Dragon’s cargo was unloaded from the capsule and our samples along with other payload items were placed into a large container. This container was first removed from the docked ship, then the empty Dragon capsule was covered with a tarp and lifted off the boat. The exterior surface of the capsule looks pretty beat-up from the heat of re-entry, however the payload that is visible in the video looks pristine. The Dragon worked flawlessly for the return trip!

After being unloaded from SpaceX’s Dragon capsule, our space-grown fixed seedlings were returned frozen to Kennedy Space Center. I met with NASA specialists at the SSPF (Space Station Processing Facility) to remove our experiment from the space hardware. Our BRICs – the aluminum shoebox-sized containers holding our samples in their PDFUs (Petri Dish Fixation Units) had been placed in the fridge for a day in order to defrost so that the containers could be easily disassembled. The NASA team and myself gathered in the lab first thing in the morning to see how our seedlings fared while they were in space. This was the moment we’ve been waiting for! NASA hardware experts Susan and Michelle worked together to carefully take apart the BRIC container and then to open the PDFUs containing our samples. NASA quality control expert Jennifer observed the process, clipboard and camera in hand to document each step.

Opening the first BRIC, with five PDFUs and the HOBO datalogger visible.

Opening one of our BRICs, with five PDFUs plus the HOBO datalogger visible inside.

As the first PDFU was opened, we all leaned into the sterile hood to see the result: Success! Initial observation of our wild-type Arabidopsis plants was superb: Inside the petri dish sitting in fixative were dozens of seedlings, just as we hoped they would be. The next petri dish was identical. Fantastic! However, upon opening the third PDFU, things became more complicated. This was a PDFU with some of our mutant seedlings in it. These plants had also grown well, however, there were a couple of contaminating microbial colonies about the diameter of a pea that were growing with the seedlings, definitely not what we wanted to see.

We unpacked the rest of the space flight PDFUs and then moved on to the Earth-grown control petri plates. These samples are every bit as important as the space grown ones because it is vital to compare parallel samples grown on Earth. Given the contamination in the space-grown mutant we expected the same for the Earth-grown controls because we used the same batch of seeds and sample preparation protocols. However, all the Earth-grown petri plates (wild type and mutant) were pristine and filled with dozens of seedlings. Germination was excellent, the seedlings were well preserved, and there was plenty of plant material for analysis of gene expression.

space arabidopsis

Arabidopsis seedlings grown in microgravity.

Of course we are now working hard to define exactly how that contamination crept in to a few of our flight samples. Brian Hudelson, Director of the University of Wisconsin Plant Disease Diagnostics Clinic, very kindly identified it for us and no, nothing exotic or exciting, just regular old Penicillium, probably the most common contaminant found in experiments performed anywhere on Earth. We are now chasing down leads on how a few fungal spores may have slipped in as we assembled some of our sample dishes. Solving these kinds of technical problems is just part and parcel of doing business in space and is how we get better and better at designing for our next flight experiment – and yes, the Gilroy lab is going back to the ISS for another experiment! More about this in a future post.

Fortunately, all of our wild type samples (both space grown and Earth grown controls) were perfect. These are the critical samples for our analysis of gene expression and thus we will be able to answer most of the questions we set out to address with this experiment. Won-Gyu has already measured the seedlings, separated root and shoot, and isolated high-quality RNA from both samples grown in microgravity and grown on the Earth. The next step will be to quantify the changes in the expression of specific genes, in particular we are interested in what changes there might be in the expression of genes known to be regulated by low oxygen. Then using RNAseq, we will look at the shifts in the expression of all genes in the Arabidopsis genome. This technique will allow us to see what patterns of changes occur in all expressed genes and enable the discovery of other key genes that plants rely on as they adapt to growth in microgravity.

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BRIC-17 On-Orbit

As I type this, the BRIC-17 plant biology experiments are in microgravity, orbiting the earth on the International Space Station. BRICs A and B contain plant cell cultures from the University of Florida, BRICs C and D contain our Arabidopsis seedlings for the Gilroy Lab TOAST experiment from the University of Wisconsin.

577px-ISS_Expedition_34

Mission patch for NASA’s Expedition 34, the mission running currently that includes our BRIC-17 plant biology experiment.

NASA has been fantastic about keeping us updated via email as to the astronaut’s on-orbit activities concerning our BRIC-17 experiments. A few minutes ago, we just received an email from NASA:

> I bring to you more good news.  This morning, on-orbit, Dr. Simon Gilroy’s
> Canisters C and D successfully actuated at approximately 7:57AM Eastern.
> Also, earlier Canisters A and B were transferred into MELFI 1, Dewar 3 at
> approximately 3:50AM Eastern.

This report means that the fixative solution (RNAlater) was successfully injected by astronaut Marshburn into our 10 petri plates containing our microgravity-grown seedlings. We are glad to hear that all went well with the fixative injection because a test of the BRICs a week before launch indicated there could be issues with actuation. After a day at room temperature to allow the fixative to work, our two BRICs will be placed into the MELFI (Minus Eighty-degree Laboratory Freezer for ISS) by space station commander astronaut Ford until they can be packed into a cold bag for the return trip to Earth in the SpaceX Dragon capsule (which is still docked to the space station).

MELFI1

The Minus Eighty Lab Freezer on the ISS (MELFI; image from NASA).

A minus-eighty freezer is standard equipment required for most life science research. The Gilroy Lab has an upright minus-eighty in the hallway just outside of our lab to store our bacteria stocks, concentrated solutions, some dry chemicals, and frozen samples. It is heavy, has a loud compressor, a huge footprint, and is an energy hog; all these things would cause problems on the ISS. I was curious to see what the ISS minus-eighty freezer was like. Needless to say the MELFI is a high-tech marvel of engineering, if you want to read more about it NASA has a good description of the MELFI.

MELFI3astronauts

Astronauts packing samples into the MELFI. Gloves are needed to handle samples due to the low temperature (NASA).

Our BRICs need a minimum of 4 days in the MELFI to be sure that they are completely chilled down for packing into the cold bag. When the Dragon capsule had a delay in docking just over a week ago, there was some concern that if berthing was more than a couple days late then our BRICs would not have enough time in the MELFI at the end of the experiment. This would’ve necessitated cutting our experiment by a day, which means one less day of growth in microgravity and thus smaller plants. The good thing is that even such a long delay would’ve required only minimal adaptation to our experimental design. We simply would’ve analysed smaller plants, perhaps combining shoot and root for the RNA isolation instead of separating the tissue for analysis. Luckily, docking was delayed by only a day so our plants were able to grow for 8 days in microgravity as originally planned.

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NASA Write-up of Our Experiment

Bob Granath at NASA has posted a write-up of our TOAST and the other BRIC-17 experiment. He did a great job summarizing our goals in sending Arabidopsis to the space station to study plants grown in microgravity. There are also some good pictures of the space hardware that is holding our petri plates right now inside the space station, with an identical set of BRICs in the International Space Station Environmental Simulator (ISSES) at Kennedy Space Center as the ground control.

Experiment Canisters Aid in Helping Study Plant Growth in Space

728491main_bric2_XL

A Biological Research in Canisters experiment package with five Petri dish fixation units (PDFU) installed. The PDFUs each contain a Petri dish with the biological sample to be flown in space. (NASA)

Another NASA article details the launch and berthing of SpaceX’s Dragon capsule holding our BRICs. After the successful Friday morning launch powered by SpaceX’s Falcon 9 rocket and release of the Dragon capsule in low earth orbit, a nail-biting hours long session occurred as SpaceX attempted to get Dragon’s thrusters working. The thrusters are needed to get the capsule close enough to the space station so that the capsule can be grappled by the Canada arm.

loading-space-x

The cold bag with our BRICs inside being weighed prior to stowage in the Dragon capsule. (NASA)

The BRIC-17 teams had a hastily called meeting Friday afternoon when the thruster problem became apparent.  We discussed the scientific ramifications of a delay in berthing with the space station. Potentially, the delay could have been up to 2.5 days, which would have shortened the growth time of our plants in microgravity by two days to allow enough time in the freezer before returning to earth. Luckily, SpaceX was able to get the thrusters working and Dragon successfully berthed to the space station on Sunday morning, only a day late, thus allowing our experiment to run for the originally planned span of time.

SpaceX’s live feed: “Happy Berth Day!”

The Dragon capsule is scheduled for splashdown back on Earth on March 25, with return of our space-flown BRICs on March 29. Let’s hope everything continues to go well and we can de-integrate our samples from the BRICs for analysis in April!

launch 5

Falcon 9 SpaceX CRS-2 Launch, © SpaceX, Ben Cooper

PS. As promised, here is the footage from the NASA press conference which Simon participated in the day before the launch.

Categories: Plants in Microgravity | 1 Comment

SpaceX CRS-2 Launch!

In a few hours, the Gilroy lab BRIC-17 team will be at the OSB II (Operations Support Building 2) at KSC to watch the launch. OSB is about three and a half miles away from the launch pad, so we hope to have a good view. We’ll be bussed there from the visitor center early in the morning. Following a launch briefing we will wait for the launch at an outside viewing area, camera in hand!

SpaceX CRS-2 Logo

SpaceX CRS-2 Logo

Unfortunately, we will be enjoying the launch after pulling an all-nighter in order to integrate our ground controls into the BRICs with the help of NASA hardware experts. The timing of our experiment is such that our science has to be integrated into the space hardware from 3-5am Eastern time. Our flight BRICs were prepared early Wednesday morning; because the ground control is offset by 2 days we have to make a repeat set of BRICs now (early Friday morning).

What if the launch is cancelled? The scrub schedule is such that the next launch attempt will occur just under 24hr later. If there are two scrubs, our experiment needs to be replaced with a fresh setup. Therefore, if the launch is cancelled both Friday and Saturday, our two BRICs will be pulled from Dragon. The ground controls we set up tonight will then become the flight BRICs and loaded into the Dragon in time for the third launch attempt Sunday morning, and so Sunday morning between 3-5am Eastern time we will have to set up two more BRICs for the new ground controls.

simonwongyu

Simon and Won-Gyu in front of the NASA headquarters at KSC.

Tomorrow’s launch chance is at 80%. Everyone, please do us a huge favor and keep your fingers crossed for an on-time departure! (That means you too, Sis!)

In other news, yesterday (Thursday Feb 28) Simon participated in two news events.  The first was an interview with Jerry Hume , a reporter with the local Fox channel 13. We were told that while the transcript will be available online, the video will not because it will only be accessible to cable subscribers. It will air between 5am and noon Friday.

simoninterview

Jerry Hume interviews Simon about our BRIC-17 experiment.

The second news event was a pre-launch NASA press conference to discuss the science going up on SpaceX 2. The location of the press conference was in the NASA auditorium in the KSC press center, and for me it was a thrill to actually be sitting in a place that previously I’ve only seen on TV and online. There were five folks in front of the cameras, plus Josh the moderator:

  • Julie Robinson, program scientist, International Space Station
  • Simon Gilroy, BRIC-17 Lead Investigator,
University of Wisconsin
  • Marshall Porterfield, division director, Life and Physical Sciences
NASA Headquarters
  • Michael Johnson, Chief Technical Officer, NanoRacks
  • Michael Roberts, Research Scientist, CASIS
simonpress

Simon behind the desk at the NASA pre-launch press conference.

Simon did a great job communicating information specifically about our BRIC-17 experiment and speaking more generally about problems that living organisms have in space. He also fielded a number of questions from reporters ranging  from social media bloggers to tech reporters from the mainstream media. The video of this press conference is not yet available online, when it is I will update with a link.

Categories: Plants in Microgravity | 2 Comments

BRIC-17 Integration

With the official SpaceX launch planned for Friday March 1 at 10:10am, the NASA BRIC team has scheduled our pre-launch meetings and integration. While still in Madison, Simon participated in a “readiness review” meeting with NASA on Friday, going through the checklist of everything that will need to occur before flight. We have the “green light” to go ahead with our BRIC experiment. We also have a good idea of how timing of events this week will occur.

The three members of our Gilroy Lab team (Sarah, Simon, and Won-Gyu) arrived in Florida Sunday night and moved into a condo in Cocoa Beach, which will be our home for at least a week. Bright and early Monday morning we went to the badging station at Kennedy Space Center to get our temporary passes onto the base. After that, we were able to cross the security checkpoint and enter the Space Station Processing Facility (SSPF).

ocean

Cocoa Beach, Florida

Last year for our initial tests at KSC (the SVT and PVT), our lab was located at the Space Life Science (SLS) lab. However, it made more sense to consolidate all of the NASA research into the SSPF building.  The SSPF has extra space now that the space station is built, and many rooms have been repurposed in to labs and offices. We were concerned that the move would cause some difficulty for our flight integration, but so far the new space is as good if not better as the SLS lab.

Our lab suite is split in two with the other half in use by a group of Canadian researchers who will send up an experiment to study microflow on the ISS. When we arrived at our lab Monday morning, we ventured across the dividing line. Strangely, the Canadian half is really very much like our American side, just with a different accent. In fact the corridor where our lab is has many other labs on it, each with a research team working hard on setting up for either our flight or the next SpaceX launch in September. This is a pretty exciting place to be at the moment!

ISSentry

Entry to the SSPF.

Our office in the SSPF is upstairs in a large partitioned room. The BRIC-17 teams are on one side, with three desks for UW-Madison and another three for the University of Florida team. In the same room are other groups flying experiments on SpaceX2, including the microfluidics group from Canada and the Japanese space agency JAXA team lead by Sachiko Yano who will be flying stem cells.

simon

Simon at his desk in the SSPF.

Tonight (Tuesday Feb 26) we have a pre-integration meeting with the NASA folks who will be packing our experiment into the space hardware (the petri dish fixation unit, PDFU). The meeting is scheduled for 10:30pm because integration of BRIC-17 science into the PDFU will occur from 1am to 3am. We have been advised to bring plenty of food, because neither the cafes in the SSPF nor any nearby restaurants will be open.

Following integration, our BRICs will be stored in the refrigerator. Our seeds will be on nutrient gel, so it will be important that they be kept cool to prevent germination until their arrival in microgravity. Then, on Wednesday afternoon the loaded BRICs will be placed into a cold bag and moved to the SpaceX loading area. They should be on board the SpaceX Dragon capsule on Thursday in time for the Friday morning launch. The Dragon will dock with the space station on Saturday March 2, and our experiment will run from March 2 until the astronaut adds the RNAlater fixative to the samples on March 9 at 7:40pm. At 8pm the following day, the astronaut will place our BRICs into the freezer (MELFI) until landing back on Earth on March 25. The BRICs will be in the freezer at KSC until we can return to de-integrate and retrieve our samples in early April.

wongyuplanting

Won-Gyu planting seeds onto nutrient gel on Tuesday afternoon to prepare our ISS experiment.

The ground control will be offset by 2 days, so that KSC can get the on-board conditions (temperature) in order to program the growth chamber (the Orbital Environmental Simulator, OES) to mimic the space station conditions. Thus the schedule for set-up and running the ground control will be identical to the flown experiment, except everything will occur 2 days later. So we will have another all-nighter starting Thursday night to finish ground control science integration early Friday morning. We should be done in time to go watch the launch at 10:10am.

SpaceX and NASA have also established a “scrub schedule.” That is, if SpaceX does not launch on March 1 10:10am for some reason, then next launch attempt will be just slightly less than 24hr later (i.e. March 2 9:47am; if that second attempt is scrubbed then the next try will be March 3 9:24am, etc.). Our experiment is good for 2 scrubs; if two scrubs occur our ground control becomes our flight BRICs. Therefore, we will need to re-do our setup every other day until launch. Let’s hope all goes well and SpaceX launches on schedule March 1!

Categories: Plants in Microgravity | 3 Comments

Botany experiment will try out zero gravity aboard space station

Last month, Simon was interviewed about our space station experiment by Chris Barncard of University Communications. I think Chris has put out an entertaining write-up, have a look and see if you agree!

University of Wisconsin’s news report about the Gilroy Lab experiment

University of Wisconsin - Madison

University of Wisconsin – Madison

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