The use of regolith on Mars with high levels of hydrated sulfate minerals for bioregenerative life support systems will lead to exposure of plant roots to supra-optimal concentrations of both Mg2+
ions in the soil solution. The results in this study show that knockout mutant lines of the known genes in Arabidopsis encoding root plasma membrane based uptake transporters of Mg2+
ions failed to confer an adaptive advantage to a MgSO4
O enriched environment. Because sel1-10
mutants are known to take up 80% less sulfate than their wildtype background Ws, these results mean that phytotoxic effects of magnesium sulfate on overall plant growth are dominated by the Mg2+
cation. Overexpression of AtMRS2-10
) in Nicotiana benthamiana
led to increased accumulation of magnesium (Mg), manganese (Mn), and iron (Fe) per unit dry weight and per plant compared to wildtype plants 
. However, the potential role of AtMRS2-10 as a major uptake transporter of Mg2+
in Arabidopsis remains uncertain based on the observation that mrs2-10
plants did not show a significant difference in leaf Mg levels compared to Col-0 under standard nutrient conditions. This could explain why, despite the indication that Mg dominantly affects overall plant growth, the mrs2-10
mutant did not show a significant improvement in growth performance compared to Col-0. Interestingly, the dominant role of Mg is also supported by the relative tolerance of the cax1-1
transport mutant plants to elevated levels of MgSO4
O. The soil data shown in and are in line with the relative tolerance of cax1-1
on agar to high concentrations of dissolved MgCl2
, a mineral in which the Mg2+
cation is the dominant phytotoxic factor 
. In addition, Bradshaw et al (2005) observed that cax1
mutants are better able to tolerate the low Ca
Mg ratios characteristic of terrestrial serpentine soils. The CAX1 knockout mutation does not fully eliminate the effects of high magnesium sulfate on plant growth performance; the absolute FW shoot biomass of cax1-1
grown on soil for four weeks was still low (20%) compared to untreated Col-0.
The primary Arabidopsis Col-0 root transcriptome responses to elevated levels of magnesium sulfate suggest a number of candidate genes that could play a role in enhancing the growth performance of plants exposed to this nutrient stress. Transcriptome responses of Arabidopsis Col-0 roots exposed to MgSO4
O versus a control solution for 45 min. revealed over 300 differentially expressed genes. Genes of known functional category include those encoding enzymes involved in hormone metabolism, transcription factors, calcium-binding proteins, kinases, disease resistance proteins, cell wall related proteins, and membrane-based transporters. The biological significance of this seemingly diverse set of gene classes illustrates the comprehensive nature of a primary abiotic stress response. The differentially expressed genes at Time 45 encoding enzymes in the ABA, ethylene and jasmonate biosynthesis pathways point to possible changes in the synthesis of these phytohormones. ABA, ethylene, and jasmonic acid have all been implicated in adaptive mechanisms of osmotic stress, ion-mediated signal transduction and the regulation of transporters 
. The exact role of many of the differentially expressed genes at Time 45 encoding transcription factors is currently unknown, although some are associated with tolerance to other abiotic stresses. The up-regulation at Time 45 of the gene encoding ZAT7 is for example in line with the finding that ZAT7 renders plants more tolerant to salinity stress when constitutively expressed 
. The down-regulation at Time 45 of the gene encoding WRKY70 correlates with the observation that WRKY70 functions as a negative regulator of developmental senescence and is involved in plant defense signaling pathways 
. The majority of transcripts encoding kinases that are differentially expressed at Time 45 encode uncharacterized protein kinases as well as receptor-like protein kinases (RLKs) with extracytoplasmic leucine-rich repeats (LRRs) or with an extracellular lectin-like domain. RLKs are membrane-spanning proteins with a predicted signal sequence and a cytoplasmic kinase domain that have been implicated in a wide range of signal transduction pathways 
. The up-regulated expression of the gene encoding CBL-interacting protein kinase CIPK9 at Time 45 is consonant with the fact that CIPK9 is required for low-potassium tolerance in Arabidopsis 
. Genes encoding proteins that belong to the transcription factor or kinase categories might be transcriptional or post-translational regulators of transporters 
Within the group of genes encoding membrane based transporters, the Ca2+
antiporter CAX1 is one of the few transporter genes that are already differentially expressed at Time 45. Its down-regulated expression is in line with our finding that the cax1-1
mutant performed better compared to wildtype when grown on soil with elevated levels of magnesium sulfate. The slightly up-regulated expression of the gene encoding magnesium transporter MRS2-10 at Time 90 and 180 agrees with the observation that a knockout mutant of AtMRS2-10
) does not show improved growth in the form of higher FW shoot biomass compared to Col-0 at elevated levels of magnesium sulfate. MRS2 transporters have been shown to transport other ions in addition to Mg2+ 
. Schock et al. (2002) speculate that, in line with the large number of AtMRS2
family members, the function of the AtMRS2
gene family may be the maintenance of metal ion homeostasis in different cellular compartments (i.e. over different cellular membrane systems). The expression of the gene encoding SULTR1;2, a high-affinity sulfate transporter, showed no significant differences at the three time points. The fact that expression of the gene is not down-regulated in the early adaptation response of Arabidopsis roots to high levels of magnesium sulfate supports the outcome of the FW shoot biomass comparison between the AtSULTR1;2
knockout mutant sel1-10
and Col-0, which showed no advantage for sel1-10
. The gene encoding EIL3, one of the known transcriptional regulators of SULTR1;2 
, did not show a significant difference in expression at any of the time points either.
Interestingly, two sulfate transporter genes of the same family as SULTR1;2
show down-regulated expression; the gene encoding SULTR3;4 at Time 90 and 180, and the gene encoding SULTR3;1 at Time 180. The spatial and subcellular localization of these transporters is not known, and so far no influence on the expression of Group 3 sulfate transporters by the sulfur status of plants has been reported 
. This study shows for the first time that genes encoding Group 3 sulfate transporters SULTR3;1 and SULTR3;4 are differentially expressed in roots upon exposure to high levels of sulfate. The expression of the gene encoding another sulfate transporter - the tonoplast-localized H+
cotransporter SULTR4;1 - is also down-regulated at Time 180, pointing at the possible retention of excess SO42−
in the vacuole. Similarly, the up-regulated expression of the gene encoding the vacuolar MHX transporter indicates the possible storage of excess Mg2+
in the vacuole. Both MHX
could be seen as marker genes for excess magnesium sulfate recognition by the plant, since vacuolar storage of excess ions is a well-known defense mechanism against ion stress.
Several members of the CAX
(cation exchanger) family show down-regulated expression in response to elevated magnesium sulfate. The Ca2+
antiporter CAX1, which shows down-regulated expression across the time points, is localized to the tonoplast and responsible for 50% of the Ca2+
antiport activity there 
. CAX2 has been shown to transport Ca2+
into the vacuole in Arabidopsis and other plant species 
, and CAX3 is known as a weak Ca2+
vacuolar transporter. CAX1 and CAX3 can be combined as “hetero-CAX” complexes to form functional transporters with distinct transport properties 
. The down-regulated expression of the genes encoding these three vacuolar Ca2+
antiporters indicates a possible shortage of calcium in the cytosol upon high magnesium sulfate exposure.
In total, over 200 differentially expressed genes encoding membrane-based transporters were identified across the Col-0 time series. Among the genes encoding transporters of known and unknown function, candidates for improving plant tolerance to high magnesium sulfate may be found. Transporters of unknown function include for example major intrinsic proteins, cyclic nucleotide gated channels, integral membrane proteins, cation efflux family proteins, ATPase E1–E2 type family proteins, MATE efflux family proteins, transporter-related proteins and putative transporters.
The differential expression of only four unique transcripts between cax1-1 and Col-0 plants after 180 min. of MgSO4·7H2O treatment indicates that the root transcriptome responses are virtually identical for the two genotypes at this time after initiation of treatment. In our transcriptome study we see down-regulation of the gene encoding vacuolar sulfate efflux transporter SULTR4;1 at Time 180, and up-regulation of the gene encoding vacuolar magnesium influx transporter MHX at Time 90 and Time 180 in Col-0 exposed to elevated magnesium sulfate. Both of these responses point to storage of excess magnesium and sulfate ions in the vacuole. This means that the profile is most probably not performed at a time point before plants recognize excess magnesium sulfate, especially since over 3000 differentially expressed genes were detected in Col-0 at Time 180, in addition to the vacuolar marker genes. We could therefore not foresee that after 180 minutes of exposure there were no more than four differentially expressed transcripts between the cax1-1 mutant and Col-0. One of the main reasons for the limited number of differentially regulated transcripts between the genotypes at this time after initiation of treatment may be that CAX1 is one of the few transporter genes that are already differentially expressed after 45 min. of exposure in Col-0, which means that down-regulated expression of CAX1 is an important natural response of Arabidopsis to elevated levels of magnesium sulfate. The response of CAX1 and other CAX family members in the Col-0 time series suggests that a deficiency of calcium caused by high levels of magnesium and possibly by calcium sulfate precipitation could play a major part in the reduction of plant growth performance in the presence of high magnesium sulfate concentrations. The difference between the immediately down-regulated level of CAX1 in Col-0 and the absence of CAX1 in the cax1-1 knockout mutant is apparently not sufficiently large at 180 min. after initiation of treatment to reveal many of the downstream molecular processes eventually leading to enhanced growth performance and reduced leaf Mg content of cax1-1 compared to Col-0 after several weeks of growth. This result is significant in that follow-up studies contrasting cax1-1 with Col-0 can look at profiles after possibly even days of exposure.
Conclusions and perspectives
It was initially thought that the mrs2-10 and sel1-10 mutants would have an advantage over wildtype in elevated magnesium sulfate soils, but this was not the case. However, fresh weight shoot biomass analyses showed that although mrs2-10 and sel1-10 mutants were not more successful than wildtype in this soil environment, the cax1 mutants were. Thus, cax1 mutants are currently the only confirmed genotype with partial tolerance to the phytotoxic levels of magnesium sulfate expected for the regolith on Mars.
To further characterize the response of Arabidopsis to Mars-like levels of magnesium sulfate a set of experiments was conducted to evaluate patterns of gene expression in wildtype (Col-0) and cax1 mutants. The 325 genes differentially expressed in Col-0 roots after 45 min. of exposure to high magnesium sulfate can be seen as candidate genes for enhanced tolerance. This set of genes included CAX1, 18 genes previously associated with enhanced tolerance to broad ranges of abiotic stresses, and several well-characterized genes known to enhance tolerance to other salts like NaCl. Many of the differentially regulated genes contain promoter sequence motifs that are known to play a role in ABA-mediated regulation, such as ABRE-CE. This abundance suggests that ABA plays a role in regulating the stress response to elevated magnesium sulfate. In addition, a rich pool of candidates is found in the 200+ transporter genes that are differentially expressed in Col-0 across the time points. Many genes in this set encode transporters of unknown function. These unknown transporter genes, along with the four differentially regulated transcripts between cax1-1 and Col-0, are of particular interest as they encode proteins of unknown function whose roles are now at least implicated.
This study functions as a solid basis for the development of Mars soil-compatible plants by reducing the number of potential candidate genes from tens of thousands to several hundred. Future research efforts could determine whether any of these candidate genes, or their potential regulators, can be confirmed as genomic loci for (crop) plant growth enhancement in the presence of mars-like levels of magnesium sulfate.