EXPERIMENT RECORD N° 9834
Canes - Effects of microgravity and space environment on ligneous plant canes
  1. 2020 • ISS Increment 63
  2. 2020 • ISS Increment 64
Life Sciences:
  • Plant Biology and Physiology
S. Cluzet (1), J. Valls (1), G. Da Costa (1), A. Palos-Pinto (1), T. Richard (3), J.M. Mérillon (1), P. Darriet (1), M. Lebert (2)
(1)  
ISVV - Institut des Sciences de la Vigne et du Vin
210, chemin de Leysotte
CS 50008
33882 Villenave d´Ornon
FRANCE
(2)  
Space Biology Unlimited SAS
24 Cours de l´Intendance
33000 Bordeaux
FRANCE
(3)  
Friedrich-Alexander University Erlangen-Nuremberg
Department of Biology
Cell Biology Division: Gravitational Biology Group
Staudtstraße 5
91058 Erlangen
GERMANY
BACKGROUND
One of the most important types of raw materials in our contemporary diet are ligneous plants. This specific kind of plant has already been extensively studied from an agronomic, chemical, biochemical, and microbiological standpoint. Their characteristics, such as the variety, the terroir and the environmental conditions they experienced during the growing season are highly influencing the resulting fruits.

Due to their complexity and their unpredictability, the impact of these "environmental" parameters is very difficult to predict. It is extremely important to obtain more in-depth knowledge of the capacity of the plants to respond to environmental variations, like those we are starting to experience on Earth due to the climate change.

Thus, the science team proposes to develop a project, called Canes, capable of analyzing the responses of ligneous plant cells to a major environmental stress that is totally new for the plant, from an evolutionary standpoint.

Numerous experiments involving the growth of plants and seeds in altered gravity have been conducted in the past. This scientific area has also recently received more attention with the perspectives of very long duration orbital flights, in particular future human exploration of Mars. The study of the behavior of ligneous plants in the ISS can help us to progress in different fields of research.

Polyphenols are ubiquitous plant compounds. They can be divided in two classes: flavonoids and non-flavonoids ones. The latter group contains stilbene molecules. In ligneous plants, stilbenes can be found in all parts of the plant and they display several biological functions and mainly participate to plant defense responses to face pathogens. The basic unit is resveratrol and from this monomeric compound, other molecules can be formed thanks to glycosylation (piceid), methylation (pterostilbene) or oligomerization (viniferins, etc), as instance.

The MIB axis of the Oenology RU at ISVV studies the ligneous plants polyphenols. The researchers of this group are interesting in phenolic composition and identification (phytochemistry), and biological activities of polyphenols in plant and human health.
Thanks to liquid chromatography and/or nuclear magnetic resonance spectroscopy, the identification and the content of cane polyphenols can be investigated, as reported by Lambert et al. [2013] The major compounds identified were ɛ-viniferin, resveratrol, piceatannol, and vitisin B and, hopeaphenol and miyabenol C. The proportions between these major stilbene compounds vary as their abundance and order of abundance varied according to the cultivar. It can be hypothesized that the kind of cultivar, so the genetic of the variety, is an important point triggering that variation but it is not the single one. Indeed, different biotic and abiotic factors can influence significantly the quality and quantity of polyphenols (Andelković et al., [2015]; Chacón et al., [2009]; Taware et al., [2010]). 

Stilbenes, as resveratrol, are well-known to exhibit a large range of biological properties in plant health field (e.g. antimicrobial and anti-insect activities) and in human field (as antiinflammatory, neuroprotective, and antiviral activities). These molecules are considered as the phytoalexins of grapevine plants as they display antimicrobial activities and can be induced upon stresses.

Downy mildew consists in the most devastating diseases worldwide, causing serious economic losses in major agricultural plants such as tobacco, lettuce, cucurbits, corn, grasses or grape. Considering environment changes (rising concentrations of CO2 and other greenhouse gases, increase in global temperature and longer seasons), it is hypothesized that these conditions could increase pathogen survival, indirectly affecting disease incidence of downy mildews. In order to secure plant harvest, large quantities of agrochemicals are used to control the spread of pathogens and synthetic fungicides with adverse effects are sprayed on the environment. Chemical products with a good bioavailability can be efficient at low doses, but they generally suffer from problems of resistance. Moreover, these chemicals contribute significantly to environmental pollution and are a threat to human health. Among the promising alternative strategies of plant protection, the induction of plant natural resistance, by the use of natural plant extracts or of mildew resistant varieties, were proposed. Under microgravity (<10 g), Tuominen et al. [2009] and Millar and Kiss [2013] showed that plants have increased accumulation of secondary metabolites. Under microgravity (<10 g), plants have shown increased accumulation of secondary metabolites (Tuominen LK, Levine LH, Musgrave ME [2009]). Because secondary metabolites as polyphenols or alkaloids are plant defensive compounds displaying antimicrobial properties, plants with stronger amount of secondary metabolites are more resistant to biotic and abiotic stresses.

The Canes experiment will be conducted using ligneous plant prunings, to monitor plant responses to unfamiliar environmental conditions. Pruning canes correspond to mature shoot with dormant buds.

The science team would like to study the prunings´ capacity to grow into a viable plant under Earth conditions, after having underwent a relatively long storage period in the ISS and compare it to that of prunings which have stayed on Earth.

This will provide data on possible physiological and/or metabolic changes following relatively long-term storage on the ISS. This experiment is designed to answer the ultimate question of the possibility of growing ligneous plant under space conditions.

The subject of the Canes experiment will be ligneous plant of two types: Merlot and Cabernet Sauvignon. The experiment material will be pruning canes of the year, which are mature shoots with dormant buds. The specificity of these organs is that they are alive but in a hibernation mode so that they do not need any nutrient supplementation or watering, only an important humidity (70/80%) with a relative low temperature (between 0.5 to 8 °C) in the dark. These conditions correspond to the way of storing pruning canes on Earth in order to obtain grapevine cuttings (growing plants).

During their storage inside the ISS, the canes will be exposed to radiations (received naturally by ISS) and microgravity, during a relatively long-term period (about 4-6 months).

OBJECTIVES
The main goal of the Canes experiment is to recover canes which underwent modifications due to their storage in microgravity in an environment where more radiations are received than on Earth.

Our first objective is then to study the impact of the ISS conditions on the ability of canes to be developed into a viable plant when they return back on Earth. 
Secondly, the material will be used to quantify for the first time the mutational rate as well as the epigenetic
changes in a wood plant which is considered to be a fruit tree model. 
Third, the cane (wood, leaves and fruits if possible) metabolism will be observed to assess the composition of several primary and secondary metabolites. Will the production of anthocyanin (natural food coloring) and stilbenes (anti-microbial compounds) vary?

In addition to gather new information concerning plant responses to new environmental conditions, this project may lead to obtain new biologically-active products. For example, the plant cells may produce compounds that in the on-Earth conditions could not be identified, due to their slight content and/or to their absence, but that could be noted thanks to specific reactions triggered by the space environment. These potential new products could be of interest for food industry, pharmaceutical and cosmetic purposes due to hypothetical new anti-radiation and/or antioxidant properties.

SPECIFIC GOALS and DETAILED OBJECTIVES
The experiment done with ligneous plant canes in the ISS environment will address several issues:
  • Even in a dormant state space radiation exposure as well as micro-gravity stress will induce mutations in the canes as well as epigenetic modifications.
The in-depth genetic analysis of buds and stems will supply a first time quantification of the mutational rate. These quantifications are also required for experimental estimates in future missions.
  • Is a ligneous plant organ, placed after a long-stay (several months) in space conditions (microgravity, radiations, low temperatures, stems, leaves, berries, if possible)?
It will help scientists to understand the influence of specific parameters (as microgravity or radiations) that enables a correct plant development. Moreover, it is planned to use two different ligneous plants varieties in order to gain information about their ability to respond to ISS conditions. It could help to select plants with better potential to respond to harsh environment.
  • Do we find metabolic modifications in these living organs? At which levels? In the cane wood? In the leaves of rooting canes which have been previously stored during long periods in the ISS?
It will help to understand the impact of specific parameters (as microgravity and radiations) in the synthesis of metabolites, polyphenols as instance.
  • If the ISS environment has triggered variation (quantitative and/or qualitative) in the metabolite composition: are the plants able to differentially respond to stresses, such as pathogens or elicitors? It will give us information about the potential key elements implicated in defense responses.
Does ISS environment enable the plants to produce compounds with enhanced quality? Do these metabolic variations allow obtaining plant extracts/molecules with enhanced biological activities?

For instance, the ISS cane extracts could display stronger biological property as antimicrobial and/or anti-oxidant ones. If their biological properties are enhanced, new strategies, potential strategies allowing to mimic space conditions, could be proposed in order to develop such enriched natural extracts to be used for cosmetic or nutraceutical concerns.

JUSTIFICATION FOR SPACE EXPERIMENT
In order to carry out the Canes objectives, we have selected among the different ligneous plants available grapevine (Vitis vinifera L.) as it is considered to be one of the major fruit crop worldwide. Grapevine is also a plant rich in polyphenols and particularly in stilbenoids.
These compounds share a common unit, the trans-resveratrol from which many more or less complex compounds can be produced in the plant. Wood is one of the organs which contains the most qualitative and quantitative content of complex stilbenoids. Finally, this plant is a vivacious species. Assessment and improvement of the resistance durability of vivacious plants are crucial issues, knowing that the introduction of resistance genes in a new variety is a long-term and costly process.

The plant material will be for most of the experimental time in a dormant state. This state is not comparable to seeds, which were in many previous experiments exposed to hard UV and solar radiation, space temperatures and vacuum and resulted in nearly no detectable changes in the plant germination rate or growth.

The originality of this study is to perform experiments on a relatively long-term period and with a never-experienced plant material form, ligneous canes. The wood canes used are not as passive as seeds, but shows a slow metabolic activity. Working with pruning canes offers the possibility to a new way to study the impact of ISS in plants, as canes are between growing plants and "truly" dormant organs, as seeds. Indeed, with these passive growing organs, we can study for several months the ISS impact on plant metabolism. Furthermore, we planned to expose some of the canes to growing conditions few days before their return to increase our opportunities to get a new plant material.

The ISS environment is harsh regarding the influence of microgravity combined with radiation. In response to this environment, epigenetic changes as well as mutations are to be expected. Up to now the epigenetic changes as well as the mutational rate was never quantified in a wood plant and to our knowledge in other plants although they could trigger specific technological properties that will be evaluated following their recovery, if particular metabolic differences were noted on the ISS canes. This experiment is therefore a first timer.

The secondary components produced by vine are defense/stress responses which might well be transiently or permanently changed by the processes mentioned above.

Many experiments have been conducted in order to increase the capacity of grapevine to produce stilbenes by the use of biotic stressors (phytohormones, microbial molecules, etc) and of abiotic stress (wounding, UV radiations as instance). But as far as we know, the microgravity and ISS radiations were never tested as specific inductors of stilbene metabolism. Moreover, Tuominen et al. [2009] and Millar and Kiss [2013] have already reported that under microgravity (<10-3 g), other plant species can accumulate more secondary metabolites. For that reason, space represents a new tool to gain the opportunity to affect the stilbene production. Besides, to our knowledge no studies have been performed with ligneous plant canes and grapevine is considered as a model for fruit trees genetics.

Grapevine also offers the opportunity to work with a "simple" model as pruning canes. By this way, and thanks to the ISS environment, especially microgravity, after a relatively long storage period we intend to obtain new grapevines with enhanced polyphenol synthesis capacity and with different stilbenes in a chemical point of view. These modified plants might present enhanced resistance capacity to pathogens, as downy mildew. Thus, by using the ISS environment, we will open a new avenue of research and the possibility of achieving plants with enhanced agronomical traits that will be used in the future.

In summary, ISS conditions will enable to place living material of wood plant in the context of unusually, unfamiliar environmental conditions for a relatively long time period. The expected results will offer new data on the development of this kind of plant and new information about their ability to produce secondary metabolites in the space environment or on Earth after a relatively long storage period on the ISS.

The CANES experiment is part of the Mission WISE, a research project in partnership with CNES and ESA, focusing on the future of agriculture. https://space-cu.com/mission-wise/  
Grapevine (Vitis vinifera L.) will constitute the plant material of interest. Two varieties will be experimented: Cabernet Sauvignon and Merlot. For the study, pruning canes (mature shoots with dormant buds) will be used. (see: Figure 1) Indeed, these organs can be easily kept alive (at 4°C with a tolerance from 0.5 to 8°C with humidity and air, in darkness) for a relatively long term period. Moreover, they can easily be grown in a foliar cutting post-storage, allowing experimental analyses not only on the woody parts but also in all newly developed organs, as well as the plant development by itself.

Canes will be cut in small pieces and stored in watertight containers that allow ISS atmospheric air to pass through the canes containers. As explained hereafter, the main constraints are: 
1) to maintain thermal control within the containers, 
2) to store the canes so as not to damage buds and 
3) to allow air exchange between the inside of the containers and the ISS environment.

Apart from the thermal regulation, the containers as such, are passive. During all transport processes, the containers have to stay at 4°C (tolerance from 0.5 to 8°C) and in dark conditions. Late access (or late load to launcher) is needed in order to limit the time between the harvest and the arrival on the ISS.

The Canes experiment itself will start with launch then the storage of the canes containers in the ISS and will finish with the return of the containers back on Earth.
Canes will be placed in two equal sets:
C1 set: The first set of 95 to 224 canes will be stored at 4°C (tolerance from 0.5 to 8°C) during the whole mission duration.
C2 set: The second set of 105 to 256 canes will be stored at 4°C (tolerance from 0.5 to 8°C) first and then at ISS ambient temperature (24°C (tolerance from 15 to 30°C)), fifteen (15) days before their return to Earth.

On-board the ISS, the canes will be stored during a relatively long-term period at 4°C (with a tolerance from 0.5 to 8°C) in darkness, with a humidity rate of 70/80% and with air exchange with the station environment. During their storage in the ISS, they will receive moderate amounts of space radiations (different from the ones received on ground due to the thickness of atmosphere or the trapping of solar particles in the terrestrial magnetic field lines) and will be exposed to microgravity.

Before download to Earth, some containers must be located at ambient temperature. Short before return to Earth, all containers must be taken from the stowage location (4°C and ambient) and loaded into the return craft. 

Only two parameters are required to be recorded and provided to the science team for this experiment:
1. Temperature profile recorded by devices placed in some containers of each set
2. General assessment of ionizing radiation flux in the Columbus cabin (it is acceptable to provide this from the existing on-board radiation dosimetry measurements, such as from DOSIS-3D)

Several post-flight analyses will be performed in order to evaluate the impact of these conditions on the ability of canes to develop a viable plant after a long-period storage in ISS.
The plant metabolism (primary and secondary metabolites) will be assessed, and we will focus especially on anthocyanins (natural food coloring) and stilbenes (anti-microbial compounds). Furthermore, the science team will investigate the presence of new molecules produced by the plants under ISS environment, and not present on canes stored on ground.
These new molecules could represent potential biologically-active products for pharmaceutical and cosmetic purposes.

The needs in term of number of canes for the scientific analyses: 
A minimum of 200 canes in total is required. It is expected to upload between 200 and 480 canes. The optimal number of canes for the mission is 320.

2 sets of canes will be prepared:
95 to 224 canes, with an optimal number of 160 canes, contained in the C1 set will be stored at 4°C (tolerance from 0.5 to 8°C) for the whole duration of the experiment.

105 to 256 canes, with an optimal number of 160 canes, contained in the C2 set will be stored at 4°C (tolerance from 0.5 to 8°C) until 15 days before their return to Earth (3.5 months if the whole duration of the experiment is 4 months for instance). Then, for the last 15 days, they will be placed by the crew at ISS ambient conditions (24°C (tolerance from 15 to 30°C)).

Half samples (C1 set) have to stay at 4°C (tolerance from 0.5 to 8°C) in the dark during the travel. The other half (C2 set) has to be at 24°C (tolerance from 15 to 30°C) in the dark during the travel (see Figure 2).
It is tolerated that the C1 set stays out of cold stowage for a maximum of 72 h if it makes the download easier. However, transportation of the C1 set from landing site to Europe has to be performed at 4°C (tolerance from 0.5 to 8°C) and in the dark.
Early retrieval is required to minimize the duration during which C1 set canes are out of cold stowage facilities from stowage at 4°C in ISS until delivery at the landing site.

GROUND EXPERIMENT
A ground experiment will be run in parallel of the flight experiment. Two sets of ground containers will be established in conditions C3 and C4 and will follow the expected profile of the spaceflight containers in conditions C1 and C2.

UPLOAD to ISS
The SpaceX Dragon cargo spacecraft launched for the CRS-20 commercial resupply service mission on 7 March 2020 at 04:50 UTC and brought 320 vine plants to the ISS. After Dragon´s berthing to the Station on 9 March 2020, the vine canes were stored and will remain on board of the ISS for six months. 
PLANNED ANALYSES
As soon as the canes will be back in the ISVV lab, the canes will be separated in two batches.
One batch will allow to directly perform metabolite as well as the genetic analysis on the wood canes, and the other one will be used to follow the plant development (rooting, stem and leaves development, etc.). Moreover, on this second batch, some metabolic analyses will be also carried out at the level of the leaves, in order to be not destructive for the plant.
The following analyses will be performed: total polyphenol content measurement, quantification and identification of the stilbenes, anti-oxidant assay such as DPPH, ORAC and metal chelating assay. More analyses as epigenetic ones will be performed at ISVV lab or in other scientific institutes.

PLANNED SCHEDULE
As soon as the canes will be in the ISVV lab: analyses of the cane wood.
As soon as the canes will be in the ISVV lab, plants will be developed. Rooting generally takes at least 15-20 days, bud development with leaf expansion could occur after 1 month, a plant with 5-6 leaves will be obtained in 2 months and so analyses could be performed.

EXPECTED RESULTS and HYPOTHESIS
After their ISS storage period:
- Our main hypothesis is to recover plants with slight modified metabolites. For instance, their ability to produce some polyphenols as stilbenes could be impacted.
- We also expect to note differences in the development of the plants, as delay, or at contrary improvement, in the rooting or in the growth.
- We finally expect an increase of the mutation rate and we will quantify the epigenetic changes after extended period of space exposure.
If metabolic modifications are noted in canes, plants would develop further in order to obtain fruits. Indeed, we hope that the modifications would have affected not only some cells but the whole materials and so consecutive changes could occur at the level of the berries.
[1]  
J.L. Chacón, E. García, J. Martínez, R. Romero, S. Gómez, (2009), "Impact of the vine water status on the berry and seed phenolic composition of ´Merlot´ (Vitis vinifera L.) cultivated in a warm climate: Consequence for the style of wine", Vitis, 48, 1, DOI: https://doi.org/10.5073/vitis.2009.48.7-9, pp. 7-9.
[2]  
L.K. Tuominen, L.H. Levine, M.E. Musgrave, (2009), "Plant secondary metabolism in altered gravity", Methods in Molecular Biology, 547, DOI: 10.1007/978-1-60327-287-2_30, pp. 373-386.
[3]  
P. Taware, K. Dhumal, D. Oulkar, S. Patil, K. Banerjee, (2010), "Phenolic alterations in grape leaves, berries and wines due to foliar and cluster powdery mildew infections", International Journal of Pharmacy and Biological Sciences, 1, pp. 1-14.
[4]  
C. Lambert, T. Richard, E. Renouf, J. Bisson, P. Waffo-Teguo, L. Bordenave, N. Ollat, J.M. Mérillon, S. Cluzet, (2013), "Comparative analyses of stilbenoids in canes of major Vitis vinifera L. cultivars", The Journal of Agricultural and Food Chemistry, 61, 47, DOI: 10.1021/jf403716y, pp. 11392-11399.
[5]  
D.L. Millar, J.Z. Kiss, (2013), "Analyses of tropistic responses using metabolomics", The American Journal of Botany, 100, 1, DOI: 10.3732/ajb.1200316, pp. 79-90.
[6]  
M. Andelković, B. Radovanović, A. Milenkovic-Anđelković, V. Radovanović, (2015), "Phenolic compounds and bioactivity of healthy and infected grapevine leaf extracts from red varieties Merlot and Vranac (Vitis vinifera L.)", Plant Foods for Human Nutrition, 70, DOI: 10.1007/s11130-015-0496-3, pp. 317-323.
[7]  
I. Morales-Castilla, I. García de Cortázar-Atauri, B.I. Cook, T. Lacombe, A. Parker, C. van Leeuwen, K.A. Nicholas, E.M. Wolkovich, (2020), "Diversity buffers winegrowing regions from climate change losses", PNAS - Proceedings of the National Academy of Sciences of the USA, 117, 6, DOI: https://doi.org/10.1073/pnas.1906731117, pp. 2864-2869.
click on items to display

Figure 1: Principal structures of a grapevine.

Figure 2: Nominal timeline of the Canes experiment.

Figure 3: Timeline of the Canes experiment with a C1 download at ambient temperature.

Figure 4: Picture of four canes.

Figure 5: Detail of the preliminary design of the Canes container.

Figure 6: The CANES experiment is part of the Mission WISE, a research project in partnership with CNES and ESA, focusing on the future of agriculture. https://space-cu.com/mission-wise/
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