Microsome-associated proteome modifications of Arabidopsis seedlings grown on board the International Space Station reveal the possible effect on plants of space stresses other than microgravity.

Growing plants in space for using them in bioregenerative life support systems during long-term human spaceflights needs improvement of our knowledge in how plants can adapt to space growth conditions. In a previous study performed on board the International Space Station (GENARA A experiment STS-132) we evaluate the global changes that microgravity can exert on the membrane proteome of Arabidopsis seedlings. Here we report additional data from this space experiment, taking advantage of the availability in the EMCS of a centrifuge to evaluate the effects of cues other than microgravity on the relative distribution of membrane proteins. Among the 1484 membrane proteins quantified, 227 proteins displayed no abundance differences between µ g and 1 g in space, while their abundances significantly differed between 1 g in space and 1 g on ground. A majority of these proteins (176) were over-represented in space samples and mainly belong to families corresponding to protein synthesis, degradation, transport, lipid metabolism, or ribosomal proteins. In the remaining set of 51 proteins that were under-represented in membranes, aquaporins and chloroplastic proteins are majority. These sets of proteins clearly appear as indicators of plant physiological processes affected in space by stressful factors others than microgravity.

Envisioning the use of plants as life support for long-term human spaceflights and space exploration still requires a serious commitment in studies devoted to the knowledge of plant behavior in a spatial environment in order to determine optimal conditions for their growth and development in this unusual environment.- During evolution, terrestrial plants have adapted to the ground gravity to optimize their development on earth, but they are still able to sense changes in the gravity vector and mount appropriate responses. But, beside the lack of gravity, the spatial environment introduces new conditions that could have an effect on plant behavior, such as confined culture conditions, cosmic radiations, lack of convection, gaseous environment,- and maybe some others factors likely still not suspected. Most of these factors are different or absent on ground and thus should also be considered in the interpretation of spaceflight experiments. For instance, during the Space Shuttle mission STS-81 it was observed that seedlings grown both on the 1-g in-flight centrifuge and µ g displayed a phenotype similar to seedlings exposed to high levels of ethylene (shorter seedlings, greater root hair density, and anomalous hook formation on hypocotyls). The ethylene hypothesis could be tested on the next space experiment (STS-84) where a similar phenotype was obtained for flight-grown seedlings using the 1-g in-flight centrifuge and seedlings grown on ground and exposed to ethylene. Such high suspected levels of ethylene could be confirmed by measurements in the cabin atmosphere by NASA reporting levels as high as 1.1–1.6µl l-1 during this STS-84 mission . Similarly the group of Ferl evaluated the effects of the lack of convection-driven gas movement on space-induced hypoxia.

Recently, in the frame of GENARA-A, an experiment hosted by the International Space Station (STS-132 - ULF-4 05/14/2010), we analyzed the effect of microgravity on the membrane proteome of Arabidopsis thaliana seedlings grown under either µ g or 1 g conditions in the European Modular Cultivation System (EMCS). Using LC-MS/MS analysis and UNIPROT annotations we sorted out functional groups of proteins that were found to be significantly less or more abundant in membranes during microgravity conditions. In this previous analysis, in order to assess the effect of microgravity alone, a highly stringent filter was used: A protein was considered to be suitable for analysis only if its abundance in membranes did not significantly change between 1 g in space and 1 g on ground in the same culture conditions, i.e., EMCS growth conditions.

In contrast, in the present report, we took advantage of the presence of a centrifuge in EMCS to focus on proteins whose abundance in membranes was not dependent upon gravity, i.e., quantitative changes were not significantly different between microgravity and 1-g conditions on board ISS. This set of membranes proteins that is not responsive to a change in gravity in space was then further analyzed to evaluate possible effects of the other environmental conditions found on board ISS (radiations, gases, vibrations, lack of convection…). For this purpose the relative abundances of these proteins in membranes were compared between 1 g obtained on a centrifuge in ISS and 1 g condition achieved on Earth.

We report here that large families of proteins were affected by others parameters than gravity in space. Among the proteins whose abundances in cell membranes were not changed by microgravity in the ISS (1202 from 1484 quantified proteins with at least 2 peptides; P value > 0.05), 227 proteins were found to be significantly (P value < 0.05) either more or less abundant in cell membranes of seedlings grown under 1 g in space as compared with seedlings grown on 1-g ground (all other culture conditions being equal). Among these, more than three quarters (176 proteins) were over-represented while only one quarter (51 proteins) was under-represented in membranes (Table 1). With a few exceptions the fold-change between the two conditions was close to 2 with a maximum of about 5.

Table 1. Proteins identified by LC-MS/MS and whose abundances were significantly changed (P values < 0.05) in microsomal extracts of 12-d-old Arabidopsis seedlings grown under 1 g on board the International Space Station as compared with 1 g ground control.

A. Proteins under-represented in space conditions. Ratio (ISS 1 g / ground 1 g) < 1
UNIPROT	AGI	Ratio	Annotation
Lipid metabolism
A4GNA8	AT4G25970	0.549	phosphatidylserine decarboxylase 3
Q9SZP6	AT4G38690	0.554	1-phosphatidylinositol phosphodiesterase-related protein
Q8LDH5		0.585	endomembrane-associated protein

Figure 1 displays a repartition of these proteins grouped into functional categories using UNIPROT database annotations. In the set of 51 proteins under-represented in space conditions (Fig. 1A; Table 1A) aquaporins constituted one of the main groups and were among the more under-represented, with a mean fold-change of about 3. Transporters and chloroplastic proteins formed two other important groups. We found also in this set a few ribosomal proteins and some proteins involved in the lipid metabolism. Other proteins of this set were miscellaneous proteins with diverse (25%) or unknown (19%) functions.

Figure 1A. Repartition of proteins significantly under-represented in microsomal extracts of 12-d-old Arabidopsis seedlings grown under 1 g on board the ISS as compared with 1 g ground control. Proteins were distributed into functional categories according to UNIPROT annotations.

The main group of over-represented proteins (Fig. 1B; Table 1B), representing about 25% of this set, was associated to protein synthesis and degradation: ribosomal proteins 11%, protein synthesis 2%, proteins with a protease activity (10%), and transcription regulation 2%. Other important groups over-represented in membranes were composed of proteins involved in transport (9%), lipid metabolism (7%), and oxidoreductases (5%). Proteins associated to energy and basic metabolism (ATP synthase, Cytochrome C oxydase, TCA cycle, sugar metabolism) were also found over-represented in membranes. Interestingly, the most over-represented protein in space conditions was the UDP-galactose transporter 6 (fold-change 5.6) which has a sugar:hydrogen symporter activity. Another highly over-represented protein was a chalcone-flavonone isomerase (fold-change 4.1) which belongs to the biosynthetic pathway of flavonoids known to be involved in UV-filtration in higher plants. This suggests that flavonoids might also be involved in the response to other radiations such as cosmic rays. The large set of proteins with protease activity could also be linked with the above-mentioned stress knowing that proteolytic activities can help the cell to remove damaged/oxidized proteins. The unexpected decrease in aquaporins observed in plantlets grown in space conditions may be related to the different plant water status existing in the EMCS between the ISS and ground localization.

Figure 1B. Repartition of proteins significantly over-represented in microsomal extracts of 12-d-old Arabidopsis seedlings grown under 1 g on board the ISS as compared with 1 g ground control. Proteins were distributed into functional categories according to UNIPROT annotations.

Altogether, our results show also that in space, factors other than microgravity, may affect various aspects of plant metabolism, such as protein and lipid metabolism or transport. Interestingly, although EMCS light conditions were the same in the ISS and on ground, differences were observed in the amount of chloroplastic proteins.

More experiments would be necessary to analyze more deeply the effects of the various factors that could modulate plant physiology in space conditions. These preliminary and purely descriptive results show that beside the major effects of microgravity on plant growth and development, which can be overcome using centrifugation, other uncontrolled factors that are present in a space station (e.g., cosmic rays) may have non negligible effects on the physiology of plants and thus should be taken into considerations for long-term missions.