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Please type the verification code sent to. Remember Me. Login With WhatsApp. Resend OTP Subsequent ANOVA analyses assessed whether selected mutant lines could alleviate the growth-limiting effects seen in wildtype. Results showed that FW shoot biomass and leaf chlorophyll content of mrs and sel lines were indistinguishable from that of wildtype at the tested concentrations Fig.
To control for environmental variation, each tray was divided in 8 sections, with a mutant line and its associated wildtype background line planted in alternating fashion. Yellow boxes highlight the sections with mutant plants within each flat; the other sections contain wildtype plants. The plants shown here are 3 weeks old and were harvested and analyzed after completing 4 weeks of growth.
The experiment was repeated three times. The number of genes with a significant difference in expression of over 2-fold increased accordingly, from to and The x-axes show log2 values of the fold changes in gene expression between sample sets. Each dot represents one of transcripts. Vertical lines indicate absolute fold change values as indicated on top of the graphs. Time 45 to reveal genes involved in the metabolic response to this environment as candidate genes that could play a role in high magnesium sulfate tolerance.
The resulting set of genes with significant differences in expression was analyzed for over- and under-represented GO molecular function categories. Significantly over-represented categories were polygalacturonase activity, calcium ion binding, and transcription factor activity Table 2.
The better characterized of these genes are listed by category in Table 3. For example, phytohormone associated genes encoding enzymes involved in ABA 9- cis -epoxycarotenoid dioxygenase , ethylene 1-aminocyclopropanecarboxylate synthase and jasmonic acid lipoxygenase biosynthesis pathways show up-regulated expression. Genes encoding cell-wall and disease-resistance associated proteins are also well represented; fasciclin-like arabinogalactan proteins, UDP-glucose 4-epimerase RHD1 , xyloglucan endotransglucosylase XTH9 and beta-expansin EXPB1 show down-regulated expression, while the expression of several genes encoding alpha-expansin proteins is up-regulated at Time The bp upstream regions of the down-regulated and up-regulated genes at Time 45 were statistically analyzed for over-represented six-mer sequences motifs , which may correspond to cis-regulatory elements.
Table 4 lists the ten motifs with the highest significance lowest p-values for both the up-regulated and down-regulated subsets of genes.
The differentially expressed genes at Time 45 were furthermore compared with a cluster of genes that are known to be differentially expressed in response to a broad range of stress conditions, including cold, osmotic stress, salinity, wounding, and biotic stresses [28]. The comparison showed that at least 18 of the genes differentially expressed at Time 45 appear to be universally responsive to stress conditions Table 5.
Of special interest is the differential expression of transporter genes as they may represent a metabolic strategy for tolerance to an elevated magnesium sulfate environment. Over different genes encoding membrane-based transporters were differentially expressed across the Col-0 time series Table S2.
Since the Time 90 and comparisons are not fully controlled for diurnal effects, gene expression differences for several transporter genes of interest to this study were analyzed by Q-PCR using diurnally controlled samples.
The number of differentially expressed transporter genes increased from 13 at Time 45, to 74 at Time 90 and at Time Table 2. The expression of the unique genes encoding transporters across the Col-0 time series was analyzed by cluster algorithms to reveal subsets of genes with corresponding patterns of expression Fig. The cluster analysis is based on transporter genes with significant expression at Time 45, 90 or Yellow denotes a higher, and blue a lower expression of a gene in the treated plants versus the control.
The figure shows that distinct clusters of expression patterns can be distinguished within the group of transporter genes across the three comparisons. The full cluster set is shown on the left; subsets of the clusters are expanded to the right to allow closer inspection of the differential expression patterns.
The first 10 letters of the annotation are provided in the expanded sections. The fully annotated figure can be found in the Supplemental material Fig. The summary shows that the expression of the genes encoding magnesium transporters MRS and MRS is slightly up-regulated. Besides the genes described above, there were many examples of differentially expressed genes encoding transporters of unknown function belonging to several large transporter gene families.
Represented families include the MATE efflux family, the ABC transporter family, the integral membrane family, the major intrinsic protein family, the cation efflux family, the cation-chloride cotransporter family, the anion exchange family, and the ATPase E1—E2 type family. In addition, several genes encoding transporter-related proteins and putative transporters were differentially expressed.
The gene expression ratio distributions of transporter genes that showed significant differences in expression in the root transcriptome analysis of Col-0 treated for min.
Results show permutated expression data that are calculated by the REST statistical analysis software, which uses randomization techniques. The graphs give an impression of the expression ratio distribution per gene related to the results presented in Table 7. Only 4 unique transcripts showed a significant difference in expression between the cax and Col-0 sample sets treated for minutes Table 8. Among those, the transcript with the smallest q-values and largest differences in expression was CAX1 , the gene that was knocked out in the mutant cax , and which is represented on the microarray by two different probes.
The other three transcripts included At3g, an expressed protein with similarity to beta-galactosidases in Arabidopsis, At4g, an unknown protein with similarity to other unknown proteins in Arabidopsis, and chromosomal region CHR—, which corresponds to a portion of the mitochondrial photorespiration gene At2g This could explain why, despite the indication that Mg dominantly affects overall plant growth, the mrs mutant did not show a significant improvement in growth performance compared to Col 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.
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 [30] , [31] , [32].
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 [33]. 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 [34].
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 [35]. Genes encoding proteins that belong to the transcription factor or kinase categories might be transcriptional or post-translational regulators of transporters [37].
Its down-regulated expression is in line with our finding that the cax 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 MRS at Time 90 and agrees with the observation that a knockout mutant of AtMRS mrs does not show improved growth in the form of higher FW shoot biomass compared to Col-0 at elevated levels of magnesium sulfate.
Schock et al. 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 sel and Col-0, which showed no advantage for sel The gene encoding EIL3, one of the known transcriptional regulators of SULTR1;2 [39] , did not show a significant difference in expression at any of the time points either.
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 [40]. Both MHX and SULTR1;4 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. In total, over 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 cax and Col-0 plants after min. In our transcriptome study we see down-regulation of the gene encoding vacuolar sulfate efflux transporter SULTR4;1 at Time , and up-regulation of the gene encoding vacuolar magnesium influx transporter MHX at Time 90 and Time 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 differentially expressed genes were detected in Col-0 at Time , in addition to the vacuolar marker genes. We could therefore not foresee that after minutes of exposure there were no more than four differentially expressed transcripts between the cax mutant and Col 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.
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 cax knockout mutant is apparently not sufficiently large at min. This result is significant in that follow-up studies contrasting cax with Col-0 can look at profiles after possibly even days of exposure.
It was initially thought that the mrs and sel 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 mrs and sel 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 genes differentially expressed in Col-0 roots after 45 min. 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. This abundance suggests that ABA plays a role in regulating the stress response to elevated magnesium sulfate. Many genes in this set encode transporters of unknown function.
These unknown transporter genes, along with the four differentially regulated transcripts between cax 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. ANOVA t-test results are shown for specific genotype concentration effects in the growth experiments. The fully annotated Figure 7 : hierarchical average linkage cluster analysis of transporter gene expression using uncentered correlation.
Hierarchical average linkage cluster analysis of transporter gene expression using uncentered correlation. We would like to thank Dr. Takahashi for providing the sel line. We also wish to thank B. Laughner for assisting with the harvest of hydroponically grown Arabidopsis roots, and Dr.
Ostrow, for the microarray hybridization, scanning, and data extraction. We must also thank Dr. Benedict for assistance with the microarray data normalization, and S. Norton and J. Performed the experiments: AMV. Browse Subject Areas?
Click through the PLOS taxonomy to find articles in your field. Abstract Background Martian regolith unconsolidated surface material is a potential medium for plant growth in bioregenerative life support systems during manned missions on Mars. Introduction Long duration human missions to Mars must rely on more than just stored supplies and physico-chemical means to regenerate air and clean water.
Download: PPT. Growth Performance Measurements and Statistical Analysis Fresh weight biomass of plants grown on soil was measured by grouping and weighing whole shoots of plants per tray section.
Hydroponic Root Growth The hydroponic set-up for Arabidopsis that was used in this study is based on two previously described systems [22] , [23]. Figure 2. Overview of hydroponic Arabidopsis growth and root harvest. Table 1. Genes and related primer sequences selected for Q-PCR analysis. Figure 4. Overview of the Arabidopsis growth experiment on soil.
Figure 5. The shoot fresh weight biomass comparisons of mutant and wildtype lines grown on soil. Figure 6. Volcano plots of gene expression comparisons at distinct timepoints.
Table 2. Significantly over-represented GO molecular function categories within the set of genes with significant differences in expression at Time Table 3. Well-characterized genes with significant differences in expression at Time 45 Col-0 treated for 45 min. Table 4. Motif analysis of bp upstream region of genes with significant differences in expression at Time 45 Col-0 treated for 45 min. Table 5. Genes with significant expression differences between Col-0 treated for 45 min.
Membrane transporters Col-0 time series Of special interest is the differential expression of transporter genes as they may represent a metabolic strategy for tolerance to an elevated magnesium sulfate environment. Figure 7. Figure 8.
Whisker box plots representing gene expression ratio distributions for the Q-PCR analysis of four transporter genes. Table 6. Table 8. Conclusions and perspectives It was initially thought that the mrs and sel mutants would have an advantage over wildtype in elevated magnesium sulfate soils, but this was not the case.
Supporting Information.
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