Long duration spaceflight missions will require the development of plants adapted to the space environment. Transcriptional profiling of mRNA is a robust method for querying plant molecular responses to abiotic stresses, including spaceflight. However, changes in transcriptional abundance do not always manifest at the protein level. The Advanced Plant Experiment APEX-07 was designed to query both total mRNA (transcriptome) as well as polysome associated mRNA species (translatome). Comparison of these two populations from Arabidopsis plants grown on the International Space Stations (ISS), and ground controls provides valuable insight into post transcriptional regulation associated with adaptation to the space environment.
APEX-07 consisted of two flight experiments carried out sequentially, which were initially analyzed independently. In order to increase statistical power and identify conserved trends among the two experiments, both RNA-Seq datasets were combined and reanalyzed. We found that, in root tissues, ~32% of genes were similarly upregulated in total and polysomal mRNA, while ~ 47% were similarly upregulated in shoot tissue. Response to stress and defense responses were among the enriched categories for genes commonly upregulated in root total and polysomal fractions, while photosynthesis related genes were commonly upregulated in both fractions of the shoot tissue.
Misregulation of photosynthesis (PS) related genes has been widely reported from several different plant spaceflight experiments. These findings are not limited to light-grown shoot tissues, and have been observed in dark-grown roots as well. Interestingly, we noted that PS related genes were highly enriched in the root total mRNA yet only ~17% of these were shared with the polysome. Conversely, both total and polysomal fractions showed enrichment of PS related genes in the shoot tissue. These results suggest that the majority of PS transcripts upregulated in roots are not actively recruited to the polysome.
Another aspect of the APEX study involves utilizing image analysis software (SOAPP – simple online automated plant phenomics) to analyze phenotypic differences between ground control and spaceflight plants. In particular, we are focused on rosette characteristics such as color, size and density. We anticipate that this approach will provide a quantitative measurement of observed qualitative differences between spaceflight and control plants.

Introduction
To grow straight and upright, plants need to control actively their posture through tropic movements. Gravitropism has long been considered as a major feature of this postural control. However, mathematical modeling approaches demonstrated that the dynamics of the tropic movement and the final shape of the plants organs are actually the result of a conflicting control by (1) graviperception, that tends to curve the plants organ toward the vertical, and (2) proprioception (the sensing by the plant of its local curvatures), that tends to counteract this curving-up movement and straighten the organ [1, 2, 3, 4, 5].
Methods
Here, we will describe original experimental devices developed in our laboratory, which we use in combination with image analysis and model-associated phenotyping [6].
Results
They allow us to (1) quantify graviperceptive and proprioceptive sensitivities, and (2) identify as yet uncharacterized proprioceptive structures and cellular motors driving straightening movements. By detailing the example of the gravitropic response of Arabidopsis floral stem – which involves differential growth- and of poplar stem – which involves the formation of tension wood - to tilting we will show how such approaches can lead to new and original knowledge in plant biology. Moreover, we will explain how such approaches can benefit from space experiments.

Overall plant architecture (consisting of the number, spacing and angle of secondary root and shoot branches) determines the efficiency of crops to capture essential resources such as water and nutrients below-ground, and light above-ground. Plant architecture is critically regulated by gravitropic growth, and recently, members of the highly conserved LAZY gene family have been demonstrated to play crucial roles in regulation of branching angle, a key determinant of plant architecture. Our previous work identified a novel dominant point mutation in LAZY4, (described as lazy4D) through an EMS mutagenesis screen in the model plant, Arabidopsis. Lateral roots in the lazy4D mutant demonstrate steeper rooting, a highly desirable trait, that maximises nitrogen uptake, drought tolerance and carbon sequestration in cereal crops, making LAZY4 (and related LAZY genes) attractive targets for gene editing to engineer deeper rooting in crop plants. However, the mechanistic basis for LAZY4 dependent regulation of root angle remains uncharacterised. Using live cell bioimaging and molecular genetic techniques, we determined that lazy4D influences root growth angle in a dose-dependent manner, and further, that unlike the WT protein, lazy4D is targeted to the actin cytoskeleton, presumably for intra-organellar plastid membrane to plasma membrane trafficking required for GSA maintenance and gravitropism. Further we show that an MKK-MPK3 phosphorylation module is required for LAZY4, but not lazy4d dependent regulation of growth angle. Collectively, our work sheds novel insights into the molecular mechanisms that underpin the lazy4D (and more broadly, LAZY gene) dependent regulation of root and shoot branching angle, and an attractive locus for the targeting manipulation of root architecture.

The recent advancements in genetic sequencing technologies have made it easier and more reliable to use genetic sequencing technologies for a wide range of research areas. We are making use of these advancements to gain insight into plant root microbiomes grown with cultivation methods for future space applications, and re-evaluating the role microbes may play in resisting the harsh conditions space flight has to offer.

The plant root microbiome is known to better plant health through symbiotic effects between the bacteria and the plant roots. The root microbiome can be split into three main layers, the soil microbiome, the rhizosphere, and the endosphere. The rhizosphere makes up the surface area of the root which houses bacteria and other microbes that come in direct contact with root excretions and help facilitate nitrogen fixation and increase the availability of nutrients essential for plant health.

For this project, we are using Next Generation Sequencing to sequence the 16srRNA V3-V4 region of bacteria harvested from the rhizosphere to determine the Bacterial a-, b-diversity for the root-associated bacteria of plants that are grown in an aeroponic cultivation system. The samples will undergo periods of drought, and we will look at the effect of drought stress on plants grown from sterile seeds and non-sterile seeds. Whilst monitoring plants from sterile and non-sterile seeds which were not subjected to drought. We aim to evaluate whether the bacterial composition differentiates between treatments as well as to monitor plant stress symptoms and when they set in, whilst gaining a more complete overview of the bacterial strains which assemble in aeroponic cultivation systems in a closed water cycle.
These results could give us a better understanding of 1) root-associated bacteria in aeroponic cultivation systems, 2) identifying bacterial species that are hard to, or have not been cultured to be identified and 3) If bacterial diversity has an effect on when plant stress symptoms set in. 4) We would also gain insight into whether or not it could be beneficial to sterilize seeds meant for cultivation in space because of the prevalence or lack of beneficial and or pathogenic bacteria.

It is important to keep in mind that root microbiomes also vary from species to species. There are a great many ways plants function in the wild that could be tested and utilized with the resources space and molecular technologies have to offer.

The future of space exploration will be contingent upon the use of plants in bioregenerative life support systems. Therefore, it is important to understand the effects of microgravity and reduced gravity on plant development. Several instruments, including the Random Positioning Machine, RPM, have been developed as microgravity analogues to help supplement the information from true spaceflight experiments (Kiss et al. 2019). The RPM is one such device designed to provide an analogue for the effects of microgravity by rotating plants and other organisms in three dimensions.

In this study, we compare the results from experiments conducted in true microgravity on the International Space Station (Medina et al. 2022) with those conducted using the RPM (in the 3D clinostat mode) on the ground. Seedlings of Arabidopsis thaliana wild-type and phytochrome mutants were grown in ISS experiments and in the omnidirectional gravity on a rotating RPM on the ground. We found that the RPM treatment caused less stress in the seedlings than did true microgravity. We also report that phytochromes A and B play roles in phototropic responses to unilateral light and that these roles differ in the two gravitational environments.

Thus, we conclude that while root phototropism in unilateral red and blue differs significantly between the microgravity and omnidirectional stimuli, the RPM can serve as an effective analogue of microgravity conditions for assessment of shoot phototropism. Our results support the more general conclusion that depending on the parameters studied, the RPM can be good analogue for true spaceflight studies and can be used to support and supplement space experiments in plant biology.

Plant Gravity Perception (PGP), launched aboard the SpaceX-13, was designed to test the threshold for gravity perception in wild-type and starchless mutants. Seedlings were grown aboard the ISS on the European Modular Cultivation System with white light stimulating polar directional growth prior to centrifugation in the dark at gravity treatments ranging from 0.003-1.00G. Phenotypic measurements identified distinct response thresholds for starchless and WT roots and were supplemented with comprehensive transcriptomic analyses across the gravity spectrum for both Col-0 and pgm plants. The phenotypic analyses showed that both genotypes were able to respond to gravity eventually but that pgm mutants required a greater force than WT before responding. At low levels of gravity where only WT responded, there was an increase in transcripts associated with transmembrane transporters and lipid membrane restructuring. In plants without starch, visible gravitropism was matched with a sudden transcriptional shift at the same intensity of gravity. Genes turned on at this level include proteins involved in vacuolar acidification, cell wall remodeling, and cytoskeletal restructuring. These genes appear to be involved in starch-independent gravitropic signaling. To maximize the utility of the PGP transcriptomic datasets, every gene expressed across the replicates was fitted to both tan-1 and polynomial (ax² + bx + c) regressions of normalized gene count vs. G-level to identify genes that were activated/repressed followed by a stable expression (tan-1) or that have a linear or exponential (or combination thereof) relationship with gravity (polynomial). This analysis identified a set of master regulators (circadian clock, mitogen-activated protein kinase kinase kinases, chloroplastic heat shock components as well as a cytochrome B and phytochrome interacting factor) where expression can be fit to a tan-¹ or a 2-degree polynomial fit with respect to the environmental G-level experienced by the sample groups/replicates. Taken as a whole, the PGP experiment has allowed starch-dependent and starch-independent responses to gravity–and even some that only occur in starchless mutants–to be identified. These findings and the broader dataset hold special relevance to the variable G-levels that will be faced during disparate ventures in extraterrestrial space agriculture.

Various types of fungi have been found in all major past and present human-made space structures and will undoubtedly accompany future crewed space exploration missions [1]. Despite their prevalence, many questions about how different fungal species react to the extra-terrestrial environment when accompanying these missions remain unanswered [2]. For example, does their pathogenicity increase, making them more harmful to humans? Or, are their material bioleaching capabilities sufficient to be used for in situ resource utilization? With the advancements in analogue ground-based facilities that simulate different aspects of space environment (differential gravity, analogue regolith simulants), studies in this area can now readily fill these knowledge gaps.

We investigated different fungal species, two filamentous fungi: Aspergillus niger (19004 MUCL), Penicillium expansum (1282 DSMZ); and, two yeasts: Candida albicans (SC 5314T), and C. parapsilosis (ATCC 22019T). The filamentous fungi were grown in malt extract agar (MEA) and the yeasts in Sabouraud dextrose agar (SDA), all supplemented with up to 50% of artificial lunar (LMS-1, LHS-1) or Martian regolith (MGS-1, JEZ-1) (Space Resource Technologies, Exolith Labs). Additionally, the growth happened with exposure to simulated microgravity (SµG) (3D-clinostat, Gravity Controller Gravite®, AS ONE INTERNATIONAL, INC, Space Bio Laboratories Co., Ltd.). We then assessed their growth rate, pH and total iron concentration in the media (semi-quantitative strips), and morphology (stereo- and optical microscopy).

We found that SµG did not significantly affected fungal growth rate or morphology. However, A. niger exhibited enhanced growth on both lunar and Martian simulants, at 5% concentration, compared to controls grown without regolith. For P. expansum, growth on lunar simulants was either slightly reduced or the same as on controls. The best growth occurred on Martian simulants in both filamentous fungi, in particular on MGS-1. Microscopic structures did not appear visually different from controls. Regarding the tested yeasts, these did not show significant alterations in the presence of regoliths or exposure to SµG. Morphology was typical for both species in all the conditions tested, with C. parapsilosis exhibiting derby-shaped phenotype, and budding patterns that did not differ from the controls.

Overall, our study demonstrates for the first time the exposure of these four species to this range of artificial lunar and Martian regoliths. Moreover, the results of SµG exposure are in line with existing literature for fungal species.