APEX-05: TOAST III Experiment, 2017
The Gilroy Lab currently has NASA funding for experimentation to understand how spaceflight affects the growth of the plant Arabidopsis. Arabidopsis thaliana (also known as Mouse Ear Cress) is the “lab rat” of plant biology, with many hundreds of researchers and many thousands of research publications contributing to a deep understanding of how this plant operates under many, many conditions. Our spaceflight experiment is the fifth of NASA’s Advanced Plant Experiments (APEX-05) but the specific experiment is number 3 in our Test of the Arabidopsis Space Transcriptome (TOAST III) experiment series. The transcriptome is the level to which all the genes in an organism are being switched on or off and measuring this pattern of gene expression is a major goal for the science we will be conducting in space.
For TOAST III, we will use new space hardware for our investigations. The plants will be growing in the Veggie Facility, a plant growth chamber that uses LED-based lighting to be able to grow plants for many weeks. Indeed, the Veggie was recently used by NASA scientists to grow lettuce to a size that the astronauts could harvest their own fresh salad. In addition, we have an opportunity to observe these plants using a light microscope on board the Space Station in the Light Microscopy Module (LMM), more on this element below.
After the inevitable launch delays and rescheduling that comes with the territory of being a spaceflight researcher, our experiment looks like it will launch on December 12th, 2017, tucked inside SpaceX’s Dragon capsule as part of the CRS-13 (cargo resupply mission #13). This is a particularly momentous launch as it is the first where both the rocket booster and the Dragon capsule will be previously “flight proven” (a great way of saying re-used from a previous mission, in this case SpaceX11). Once on orbit, the Dragon should berth with the ISS about two days later and the astronauts should be unpacking our plates and getting ready for the experiment just a few days after that. The timing is perfect. We have been down to Johnson Space Center on a couple of occasions to train astronauts about how to perform our experiment and before the recent launch delays we were in the unlikely position of having our work scheduled for the tiny period when one trained astronaut (Randy Bresnik) had finished his mission and come back down and our other trained astronaut (Scott Tingle) had yet to fly up. Now, with a December 12th launch, the timing and astronaut schedules have realigned and we are thrilled to have Scott Tingle as our researcher on orbit.
So, what exactly will we be sending to the ISS and what will Scott be doing?
TOAST III builds on our and others work suggesting that in space plants experience a low oxygen stress even though the atmosphere on the ISS is like the Earth’s with approximately 21% oxygen. The mechanism behind this effect is very likely to be that in microgravity, buoyancy-driven convection (the mixing of gasses driven by warm gas rising) does not occur. On Earth such convection mixes gasses around plants and animals and so when respiring organisms use up oxygen it is resupplied by the convective mixing of the air. In space, this does not occur and so organisms can use up their local oxygen, greatly limiting how much they then have available to continue growth.
We have previously found that on Earth, a molecular pump found on the internal vacuole membrane of plant cells is involved in low oxygen (hypoxic) signaling. When plants lack this pump, called CAX2, the plant switches on its hypoxic response all the time. Normally the low oxygen response reprograms the plant’s biochemistry and growth to be able to cope better with the reduced availability of oxygen. This helps plants survive for a few days under conditions such as when the area they are growing in becomes flooded. In the plants lacking CAX2, this response is always present and the prediction is these plants which are pre-adapted to hypoxia will grow better in space.
One other characteristic of plants grown in space seems to be an increase in oxidative stress. This is the kind of damage to human biology that high antioxidant diets are trying to combat. One major source of oxidative signals and damage is an enzyme called RBOHD, thus we are also flying plants which lack this protein to see how much it contributes to the changes seen in spaceflight.
The experimental program for TOAST III involves us planting seeds onto nutrient gel in Petri dishes at Kennedy Space Center. Then, in order to prevent the seeds from just germinating, we use a special far-red light irradiation rig designed and built by Caleb Fitzgerald, one of the undergraduate engineers working in the lab. The far-red light switches on the dormancy program of the seeds and, providing we keep the seeds in the dark, they maintain dormancy for several weeks. So, we wrap the plates with seeds exposed to the far-red light in foil and pack them for flight. After the trip to orbit, the packages are opened by the astronauts and placed in the Veggie hardware, where the lights of the growth system reverse the dormancy and trigger germination. After 8 days, the astronauts harvest the plants into special tubes called Kennedy Fixation Tubes (KFT) that allow us to mix the plants with a liquid fixative designed to stop any biology. The KFTs are then stored at -80˚C in an on-orbit freezer and then returned to us in January following the Dragon capsule’s return to Earth.
After we receive the frozen samples back in Madison, we will process them and use a technique called RNAseq to measure the levels of all genes in the plant, its transcriptome. We are hoping that by comparison of the mutants to the normal (i.e. wild-type) plants we will be able to see altered patterns of response that will tell us how these various types of plants have adapted to the spaceflight environment. We have also engineered these plants with a visual reporter (a green fluorescent protein) for the RBOHD gene that will allow us to look down the microscope at the fixed, returned plants and see to a cellular level where this gene had become active, giving us a map of responsiveness across the plant body to help put our gene level data in context.
Lastly, while on orbit, we will take advantage of an opportunity to put some of our samples into the LMM. This is a microscope on board the ISS that will allow us to remotely image our plant samples. The astronauts load a plate from our experiment into the LMM but then we remotely image the samples from NASA’s Glenn Research Center in Cleveland. The plants for this element of the work are engineered with two reporters hooked up to green fluorescent protein. One reporter changes as the oxygen levels are lowered (it’s called Rap2.12 and was kindly provided to us from Joost VanDongen in Germay) and a second reporter monitors when the plants are triggering environmental stress responses (this one is called EIN2 and was a gift from Akadipta Bakshi and Brad Binder in Tennessee). These reporters will let us literally see the stress responses to spaceflight play out in real time on orbit.
BRIC-19: TOAST II & GeneLAB Experiment, 2014
The Gilroy Lab has been again fortunate to secure NASA funding for a second experiment studying the growth of Arabidopsis plants in microgravity on the International Space Station (ISS). This experiment is called BRIC-19: Test Of Arabidopsis Space Transcriptome II (TOAST II) and GeneLAB. Similar to BRIC-17, we will use the BRIC (Biological Research In Canisters) hardware with our plants growing in petri plates inside PDFU (Petri Dish Fixation Units) as we did for BRIC-17. Our experiment will launch on September 19, 2014, tucked inside SpaceX’s Dragon capsule as part of the CRS-4 (cargo resupply mission #4). The Dragon will berth with the ISS two days later on September 21, 2014, at which point the astronauts will unpack our BRICs into the ISS and our experiment will begin.
So, what exactly will we be investigating during our second foray to the ISS?
The first half of our BRIC-19 experiment is TOAST II. Just as the lack of weight on board the International Space Station causes astronauts to lose bone mass, the weightless environment causes plants to lose their supporting structures. For the plant this means they grow long and thin in space, lacking to some degree the thickened and strengthened cell walls that they use to hold themselves up on Earth. The reason the plants are stronger on Earth is that they sense the mechanical forces generated by their own weight and lay down support materials in response to these signals. In space, the signals are gone and so the plants don’t produce the support materials. As astronaut Don Pettit (who grew the famous Space Zucchini!) put it: Plants “get lazy” in space.
Part of the machinery that lets the plant sense and respond to these mechanical forces on Earth is a group of genes called the “TOUCH” genes, so named because they are switched on in response to touch. One of these genes, named TOUCH 2, or TCH2, looks to be an important hub for a lot of information processing in the plant and so we think that the product of the TCH2 gene, i.e., the TCH2 protein, is a key regulator of the plant’s ability to sense mechanical forces such as its own weight. Dr. Janet Braam’s research group in Rice University has been able to make mutant plants with forms of TCH2 that is either always “on” or always unable to trigger touch responses. Dr. Braam very generously shared these mutants with us and so we now have plants that have this master mechanical response trigger always on or off. The plan is to compare the ‘always on’ and ‘always off ‘ to a normal plant growing in space and see if activating the touch response pathway even in the mechanically “silent” world of spaceflight can help restore growth that is more like what we normally see on Earth. We will look not only at the plants’ growth but also at their transcriptomes (the expression level of every gene in the plant) to see if the growth and gene expression of have the hallmarks of being at 1 x gravity, even in the weightlessness of space.
The other half of our BRIC-19 experiment is called GeneLAB, an exciting new program in NASA where data from experiments on the International Space Station is rapidly released to the entire research community to allow as many people as possible to study the dataset for insight into how spaceflight affects biology. The Gilroy Lab has the honor of sending the first GeneLAB experiment to the ISS!
The idea behind our GeneLAB work is that many plant biologists use the “lab rat” of plant research, Arabidopsis thaliana (also known as Mouse Ear Cress), to perform their experiments in space. This is a small, extremely well studied plant which has an enormous range of tools to help dissect its functions down to the level of genes and chemicals. Arabidopsis grows naturally in many places around the world and although Arabidopsis thaliana from Poland or China is all ‘Arabidopsis’, the plants in each area have diverged a little bit from each other and so there are varieties of Arabidopsis local to each area. These varieties are called ‘ecotypes’ and each is a little different from the next. So the question we want to answer is, do the different ecotypes used by researchers respond differently to spaceflight? If they do, which ecotype you use for your experiment might be critically important! The way to test this possibility would be to grow different ecotypes on the Space Station and compare them to the same ecotypes grown under the same conditions on Earth. Our GeneLAB experiment is to investigate this idea using three commonly used ecotypes of Arabidopsis. The ecotypes are all named after where they were found and collected, so the ones we will use are named Ws (Wassilewskija, collected in Belarus), Cvi (from the Cape Verdi Islands) and Ler (Landsberg erecta, orginally from Poland). In addition we will be using the Columbia ecotype (from Columbia Missouri, USA) in our TOAST II experiment, giving us a 4-way comparison of ecotype responses. As with TOAST II, we will look at the growth of the plants and then look at the patterns of genes that are switched on and off in each ecotype in response to growing in space.
If all goes as planned, we should get our ISS-grown BRIC-19 samples for analysis following the Dragon splashdown when the capsule returns to earth from the ISS in late October, 2014.
BRIC-17: TOAST, 2013
The Gilroy Lab experiment growing plants in microgravity, called BRIC-17, flew via Space X’s Dragon/F9 to the International Space Station (ISS) in March 2013. A number of plant scientists interested in testing their hypotheses in microgravity submitted proposals to the National Aeronautics and Space Administration (NASA) in the spring of 2012, and in June 2012 we were fortunate to be selected for funding following review by a NASA grant panel.
It can be a challenge to design an ISS experiment with the highest chance of success, given the constraints of the container size to be flown and minimal level of astronaut involvement. This limited capacity necessitates careful planning to ensure the inclusion of proper controls and enough replicates for robust statistical analysis of results. Arabidopsis (thale cress, or mouse-ear cress) is the plant we will use for our experiment, due to its small size and our extensive knowledge of its physiology and genetics thanks to decades of Arabidopsis research by the plant biology community.
Low oxygen, also called hypoxia, can be a problem for life in space and is a focus of our research. Without gravity, there is no convection and so mixing of gasses can be reduced. This in turn can lead to local increases in carbon dioxide and decreases in oxygen directly next to a living plant or animal. For example, imagine if the air movement fans on the ISS stopped running. The astronaut’s exhaled air (with depleted oxygen and high carbon dioxide) could form a “balloon” around their head. Likewise, in the absence of air movement around the plant, gases could locally build up or become depleted. For the aerial portion of the plant this problem can be minimized with the use of fans, however, what about roots where forcing movement and mixing through substrate is much harder? When grown in microgravity, roots are thought to experience low oxygen stress. We wish to address two questions with our experiment. First, do plants in space really suffer from significant low oxygen stress? Secondly, can we alleviate this problem using biological counter-measures without having to resort to costly and difficult engineering-based solutions?
Gilroy Lab postdoc Won-Gyu Choi, in the course of doing other experiments, discovered that the Arabidopsis plant survives better at low oxygen levels if it lacks a particular protein. The protein is a calcium transporter, and it is involved in moving calcium ions across the membranes of a plant cell. How this missing protein enables survival of the plant at low oxygen is not well understood, and we are currently designing ground-based research to further investigate this question.
Our experiment on the ISS will compare the growth and gene expression (transcriptome) in microgravity of two varieties of wild type Arabidopsis and four varieties of Arabidopsis lacking particular calcium transport proteins. These six varieties will be grown on nutrient gel (phytagel) in small circular petri plates that fit into special containers called Petri Dish Fixation Units (PDFU); five PDFU will then be placed into NASA’s hardware called Biological Research In Canisters (BRIC). In addition to the seeds and phytagel, the PDFU also will hold the liquid “fixative” which will be added to the plants after 8 days of growth on the ISS to preserve them for analysis after return to Earth.
NASA calls our experiment BRIC-17, also known as the Test Of Arabidopsis Space Transcriptome (TOAST).
We are grateful to have the opportunity to put TOAST on the space station and hope to contribute to our knowledge and understanding of how plants cope with the challenges of growing in microgravity. It will be a interesting journey and I hope to convey the experience to you with regular blog updates about our efforts and other plant-related space science. Check back often to see our progress and results!