Project Description

What is the issue with root growth during spaceflights?

We are entering a new era of Space exploration - spaceflights are being slated to travel much further than ever before. China plans to haul Mars samples to Earth in 2030-31, followed by crewed missions to Mars in 2033. The Artemis program by the US National Aeronautics and Space Administration (NASA) plans to build an orbiting lunar Space station to support landing humans on the lunar surface by 2024. These and similar efforts encompass many technical challenges in order to be successful. Resource scarcity and sustainability is crucial in space. The cost of sending food to the International Space Station (ISS) is estimated to be USD 20,000/kg, where each crew member receives ~1.8kg of food (including packaging) per day [1]. Efficient plant cultivation is key to feeding astronauts, as well as facilitating air regeneration and boosting astronaut mental health. Plants grown in space have unfortunately been observed to be uniformly smaller than their Earth counterparts. One likely contributor to this is their lack of gravitropism, or root growth in response to gravity. The developmental patterns of plant organs are continuously amended through perception of signals from the environment, integration of these signals, and responses to external environmental stimuli. On the ground, roots typically extend downward under gravity. Without gravitational cues in space, they instead grow toward the stems and in tight coils, and fail to establish a sufficient surface area for nutrient exchange. Furthermore, the lack of gravity in space imposes secondary environmental stresses to the plant by disrupting movement of fluids and any processes influenced by convection, such as gas exchange. Stunted root growth in spaceflight translates to lesser calories for astronauts [2][3].

(Figure 1): Plant growth in orbit being slower than comparable ground controls. Root growth patterns from 8.5 day old plants from ground control (A) and flight experiment (B). Colored traces were made at each 6 hour increments for (A, B). (C) Length comparisons between GC (ground control) and FLT (flight experiment) plants (n=11). (D) Comparison of 48 hour growth patterns between GC and FLT plants. (E) Meristematic zone in roots of GC and FLt plants. (F) Elongation zone in roots of GC and FLT plants. Image adapted from [4].

What are the caveats of currently available solutions?

Previous systems, like NASA’s PONDS hydroponics system, have partially reduced the issue with root growth by increasing the circulation of water around plant roots. However, no existing solutions address the fundamental problem, which is the lack of external cues for guiding root growth downwards. Previous studies have addressed the effect of chemotropism on root growth. A spaceflight experiment conducted aboard the ISS showed that Daucus carota exhibited a positive chemotropism towards disodium phosphate, with the roots orienting growth towards the chemical; compared to the ground reference plant [5]. However, concentration of the chemical stimulant needs to be thoroughly optimized. Moreover, the effect of chemical uptake on the normal metabolic pathway and/or nutrient content of the plant is unclear.

How does gravity affect root growth?

Gravity is an essential cue for root growth, orientation and development. In vascular plants, specialized cells called statocytes are found at the center of the root cap. In the roots of monocot and dicot plants, they are the columella cells. These statocytes contain tiny starch-accumulating plastids - statoliths - which are dense enough to sediment under gravity at the bottom of the cells. This repositioning of statoliths induces a relocalization of auxin transporters (PIN proteins) at the statocyte membrane, in turn generating a lateral transport of auxin towards the lower side of the root [6][7]. Auxin, an essential plant hormone, regulates cell division, elongation, as well as differentiation in shoots (hypocotyls) and roots; and is involved in root responses to environmental cues such as gravitropism and hydropatterning. The physiological responses are exerted as a result of tightly regulated auxin influx and efflux gradients within cells of the root cap [8]. As a result of lateral auxin transport due to PIN proteins, auxins which accumulate towards the lower side of the root are oriented laterally (perpendicular to the gravity vector), and this in turn, causes inhibition of cell elongation. This consequently induces differential root growth and orientation in the direction of gravity.

(Figure 2): The root cells that respond to gravity stimuli in Arabidopsis thaliana. (A) Longitudinal primary root showing meristem, distal and central elongation zones, and maturation zone. The boxed region is the central columella region of the root cap. (B) Layers of cells important for gravitropic response - S1, S2, S3 and S4 respectively. Central columella cells (encircled) from the S1 and S2 regions play an essential role in gravity sensing. (C) Representation of a columella cell showing how amyloplasts settle at the bottom of the cells in response to gravity. Image adapted from [8].

Can we engineer magnetotropism in plant roots?

To restore directional root growth in microgravity, we propose that the existing gravitropic mechanisms be engineered to respond to an artificial cue. We aim to engineer roots to grow in the direction of magnetic field gradients: magnetotropism. We predict that filling statoliths with iron-loading proteins, like ferritin, will allow the statoliths to move in response to a magnetic gradient. For our project we designed a genetic construct that allows for ferritin to be expressed in Arabidopsis thaliana and imported into statoliths. Therefore, statolith movement in root tip columella cells can be controlled by controlling the magnetic gradient, while simulating microgravity using a clinostat. Our engineered magnetotropism depends on the ferritin’s efficient loading with iron, transit to the statolith, and relocalization in the magnetic field gradient. The first of these problems has been examined by Matsumoto et al. [10] who demonstrated that Pyrococcus furiosus ferritin (PFt) could be efficiently loaded with iron following expression in yeast. Regarding a statolith transit peptide, while none have yet been specifically shown to target statoliths (a specific type of plastid), we contacted the Li Lab at Academia Sinica, who gave us the prFB transit peptide sequence, shown to import into root leucoplasts [11]. We also plan to test five other transit peptide sequences that we identified as good candidates. As for relocalization in a magnetic field gradient, the feasibility of the experiment design is supported by the fact that iron-loaded human ferritin has been previously demonstrated to be able to relocalize on a subcellular scale upon application of a gradient magnetic field via a magnetic tip in HEK293D cells [12].

Why do we see synthetic biology as a means to better facilitate plant root growth in microgravity conditions?

Where current solutions compensate for poor plant growth by adjusting the environment, we engineer plants to be microgravity-ready utilizing cutting-edge magnetogenetics methods. This method of engineering root cells to respond to external magnetic gradients as opposed to gravitropic cues provides a safer and controllable alternative; whilst also not altering or interfering with plant nutrient uptake as in chemical stimulant methods. Our design takes advantage of recent advances in magnetogenetics and in our understanding of the molecular dynamics behind plant root tropism - tools and fundamental information that were not available when current systems were developed for the ISS. Whereas such systems emphasize changing the local environment to suit the organism, we apply synthetic biology to directly address root growth. Genetically engineering root columella cells to express ferritin that can bind to iron and position itself in the direction of an externally supplied gradient magnetic field would facilitate directing root orientation and growth parameters as desired by externally controlling the field gradient.

How is our project important for the community and the world?

Currently, astronaut food is freeze-dried, packaged, and resupplied several times a year. These conditions restrict access to fresh foods and many perishable components of a balanced diet. Despite many efforts to bridge the dietary gap, crew members on prolonged missions still experience deficiencies in areas such as omega-3 fatty acid, β-alanine and carnosine [13]; the demands of in-flight exercises and requirements of maintaining a healthy immune system make maximizing nutrition all the more essential. In-flight cultivation could provide a more sustainable, accessible source of fresh produce than current resupply missions. Therefore, our objective of producing viable, nutrient-efficient crops is key to humanity’s ambitions in space. It would support prolonged missions, extend the distance of space exploration, and provide better nutrition and health to current and future astronauts. Furthermore, according to a recent article published by the NASA Human Research Program, ISS crew often choose to spend their leisure time gardening and find the process of growing plants beneficial [14]. Root growth in microgravity impacts all astrobotany research, and it will remain a priority due to current plant experiments on the ISS, plans for prolonged manned spaceflight, and increasing international attention on space exploration. At present, plant growth in microgravity is a focus area of the Plant Biology Program at NASA. The Vegetable Production System (Veggie) project at NASA is a space garden residing at the ISS with the purpose of helping NASA study plant growth in microgravity while adding freshly cultivated food to the astronaut’s diet. Beyond the challenges of astrobotany, gravitropism affects the development of all plants on Earth. In applications such as vertical farming, imbalances between gravitropic and phototropic (light) cues cause plants to grow irregularly - affecting output and marketability of produce. Our engineering design, which can modulate plant tropisms, can serve as a first step toward addressing such issues.

Why did our team choose to work on this project?

With recent news regarding the James Webb Space Telescope and the successes of SpaceX, we were excited to start a project with applications in space. Additionally, our team wanted to focus on a project that could take advantage of the resources available through our university. We have immediate connections and proximity to the Johns Hopkins Applied Physics Laboratory, which contains labs working on plant synthetic biology as well as civil space. Our institutional strengths put us in an effective position to carry out the project, access the necessary materials, and find qualified faculty mentors. Members of our team are also experienced with plant biology—several of us have collaborated on another project that aims to genetically engineer rice for the bioproduction of useful antivirals. Working on the community project has given us familiarity with plant tissue culture as well as binary vectors and standard parts. We arrived at the root magnetotropism project idea through the marriage of these capabilities, skills, and interests.

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