Introduction
Eutrema salsugineum is an extremophyte in the Brassicaceae that enjoys its fame mostly because of its experimental and genomic similarity to Arabidopsis. Its potential as a promising model organism also stems from its ability, in the natural world, to function in saline, drought, low-N and cold/freezing conditions, and for its efficient mobilization of resources in poor or degraded soils.1 Of all the Arabidopsis relatives, indeed of all halophytic eXtremophytes, E. salsugineum has perhaps the largest research community involved at the molecular level, and at least some is known about it from many angles other than the molecular.
Formerly Thellungiella salsuginea and even more formerly Sysimbrium salsugineum, Arabidopsis salsuginea or Hesperis salsunginea, the species has also been mis-represented – even very recently – as Thellungiella halophila (which is a, for real, distinct species, endemic only to central Asia).2,3 Nevertheless, the species has survived not only identity crises imposed by systematists and molecular biologists, but 43 million years since divergence of the tribe, Eutremeae, from Camelineae, the tribe with Arabidopsis.
Although there are at least 14 accessions available for study, two have received the bulk of the attention, Yukon – from saline meadows in the southwest corner of the Yukon territory in subarctic Canada where it commonly experiences salinity, freezing and drought stress, and Shandong – from the highly saline coastal and inland soils of the temperate Shandong province of northeast China.
Next…
Ecology & physiology
E. salsugineum is considered an obligate, non-marine halophyte. It first arose in central Asia from whence it migrated to northern China and, at least once, to North America in the last 20,000 years. It is not found in Europe. In both central Asia and North America, it is found in isolated localities whereas it is now common in northern China. Interestingly, despite time and distance, the different populations have low genetic diversity.3
In North America, its range extends discontinuously from arctic Canada, through the Rocky Mountains to British Columbia, Montana and Colorado. Quite unexpectedly, in 2010, it was also found in northeastern Mexico, ca. 1600 km from the nearest other population (Colorado).4
Seeds of E. salsugineum as well as other halophytes from the Yukon region often sit in highly saline water for long periods of time, but still, they remain viable. Under normal, in situ, conditions, germination only follows a prolonged period of after-ripening, thus preventing mid-winter unpleasant surprises. As temperatures rise and rain and snow-melt reduce soil salinity, germination – which is inhibited by salt – proceeds. The factors affecting germination, dormancy and hormonal/metabolic/transcriptome characteristics have been intensively studied.5
Stress responses
Salt tolerance – Beyond germination, although high salinity impairs growth, all available E. salsugineum accessions survive and grow up to 700 mM NaCl in hydroponic culture.6 Under controlled conditions with slow and prolonged stress applications (not shocks), both Yukon and Shandong ecotypes can tolerate salinity as high as 500 mm NaCl. The physiology of this has not been extensivly studied but it seems to involve accumulation of a variety of putative “compatible osmotica” at elevated salinities. However, relative salt tolerance is independent of the concentrations of any of these. A prime example is proline which accumulates with salt or drought in growth chamber plants, but is absent in wild plants even where soils are salt crusted, possibly because of the otherwise low nutrient status of the soils.7 At both the transcriptome and the metabolome levels, in comparison with Arabidopsis, E. salsugineum appears to be pre-adapted (indifferent) to salinity and shows little response to salinization.8
Cold tolerance – Because of its ecological propensity to living in very cold regions, E. salsugineum has been used as a study system for freezing tolerance. Yukon can, in its vegetative state, survive freezing to −18°C; Shandong, on the other hand, is less adaptable. Under controlled conditions, the Yukon ecotype can more than satisfactorily complete its life cycle – from seed germination to seed production – at 5˚C.9 And even though mid growing season temperatures in the Yukon may normally be ca. 20˚C, it is not unusual for low temperatures to be 30˚ below that for short periods.10 Near freezing temperatures growth in chambers induce several effects on plant phenotype reminiscent of what plants look like in the field, including temperature-correlated leaf morphology and shifts in flowering development and phenology. Perhaps the important bit here is that growth chamber plants are not like wild ones.
The relationship between cold tolerance and photosynthesis had been convincingly demonstrated earlier. Griffith et al. 9 showed half of all the cold-repressed transcripts (using microarrays) were associated with photosynthetic processes, including chlorophyll a/b-binding proteins, plastocyanin, and PSI subunit proteins. Efficient protection of the photosynthetic machinery in plants developed at low temperature was indicated by maintenance of Fv/Fm at 4˚C while the capacity for NPQ – a major photoprotective mechanism – was altered only minimally.10
So, while the bases for the differences between the more eXtreme genotypes and those of the wimpier varieties remain unclear, for the present, it seems like E. salsugineum, or at least the Yukon ecotype, may be a useful model at the molecular level of an annual cryophyte.
Drought tolerance – In addition to cold and salt, E. salsugineum can also withstand water losses in excess of 40% of their fresh weight in growth chamber studies.10 In both genotypes, drought stress is met with a decrease in leaf solute and total water potential while turgor potential changes very little.7 Yukon and Shandong are not identical in their molecular and physiological level responses, however – Yukon is more resilient in repeated stress episodes due to differences in osmotic adjustment and other alterations in metabolism. The two genotypes also accumulate different osmotica and show different profiles in dehydrin gene expression.
In a remarkable pair of papers, Elizabeth Weretilnyk’s consortium compared growth chamber plants and plants collected in dry and wet years from a Yukon field site. In the first, microarray hybridization identified marked differential expression between field and unstressed, chamber-grown plants. There were comparatively few overlaps between genes expressed under field and cabinet conditions.8 For example,with respect to putative drought-related genes, only 20% were expressed both during a drought year in the field and in chamber drought experiments. Proline, often considered a major metabolite in stress responses, accumulated markedly in stressed cabinet-grown plants but was virtually absent in plants at the Yukon field site, even though the soil was salt encrusted. Again, growth chamber plants are not like wild ones.
With the availability of the E. salsugineum (Shandong) genome, the Yukon transcriptome, and the rapidly falling prices of sequencing, the study was extended to the whole transcriptome using RNA-seq.12 In what may be the only whole-genome comparison of the two ecotypes to date, over 39,000 SNPs distinguished the Yukon and Shandong accessions; fewer than 4,500 differentiated Yukon field plants from a lab inbred line. And contrary to their expectation that using transcriptomes from plants originating in the wild would be limited by uncontrolled genetic and environmental factors, it was not. Clearly, RNA-Seq using native plants in natural environments will be a major tool to analyze stress tolerance and the life styles of eXtremophytes.
Other edaphic stress tolerance – A number of the differences between chamber and field plants, e.g. the differences in proline accumulation under drought, probably reflect edaphic and irradiance conditions in the field7. For example, unlike the standard, unrealistic soil and nutrient conditions associated with almost all greenhouse/growth chamber studies, in the field:
- Salinity was about 10 ppt at 18 cm with salt crusted at the surface.
- Soil temperatures in root zone (ca. 18˚C) were lower – but less variable – than daytime air temperatures (ca. 24˚C); chamber pots would have little pot-to-air differences.
- Field soil had low N and P, and very high Mg, low Ca, very high sulfate, very low chloride.
- Field soil pH (8.3) was much higher than chamber soil mixture or hydroponics (ca. 5.6).
- Light intensities in the field were 7 times higher than in the chambers.
Velasco et al.1 addressed P-limitations in particular. They found constitutively high expression of the so-called “P-starvation” genes and phenological indifference to “starvation” at 0.05 mM Pi (although this would be still considered a quite high concentration in many natural soils). As is certainly common for plants in their normal ecological settings, field plants, they noted, failed to show the “classical” symptoms of P starvation.
The only consistent traits for plants exposed to variable Pi, whether as seedlings in culture or mature plants on soil, were a positively correlation in Pi content with increased Pi in the medium, and an inverse correlation with EsIPS2 (inositol-3-phosphate synthase isoenzyme 2) transcript abundance. The content vs. medium correlation shows that the plants were P-limited, and maximally responsive to availability, so perhaps the “P-starvation” genes are really P-efficiency indicators.
Another interesting result, consistent with the habitat preference of the species, was the apparently constitutive expression of several Eutrema orthologs of Arabidopsis PSI (phosphate starvation inducible) genes, indicating that the response to “P-deprivation” may be hard-wired into the regulatory network of Eutrema.
Genomics/transcriptomics
E. salsugineum has 7 chromosomes and its genome is approximately twice the size of Arabidopsis thaliana or Schrenkiella parvula. The genome of the Shandong ecotype has been sequenced twice and published.13,14 Both projects identified the major cause for the genome size difference with Arabidopsis as the ca. 50% of the genome occupied by repetitive sequences. Yang et al.13 suggested that this may reflect “stress-induced activation of TEs.” (Interestingly, the opposite conclusion was reached in studies of some resurrection plants and carnivores. 15) Other size difference are attributable to the ca. 30% greater intron length in the Eutrema.13
The first report assembled the genome to nearly complete chromosomes and included a map comparing the gene and gene block arrangements of E. salsugineum with the arrangements on the 5 chromosomes of Arabidopsis.14 The authors also quantified tandem, segmental and retrotransposon-directed duplications as well as the TE characteristics. Utilizing comparative genomics and experimental approaches, particular attention was drawn to genes related to cation transport, abscisic acid signaling, and wax production as possible contributors to its success in eXtreme environments.
The second E. salsugineum genome assembly report emphasized three major findings.13 First, the authors noted the large number of F-box genes, and that 55% of them had arisen by tandem duplication. The repetitive sequences were found to reside mostly in pericentromeric regions. Second, they identified and characterized 141 miRNAs, and the expansion of numerous stress-related species with potential targets in stress-related pathways. Finally, they examined changes in codon usage, particularly relating to ion transport genes, and suggested that the translation of those proteins may be more efficient in E. salsugineum than in Arabidopsis, and that overall, coordination of halophytic adaptations reflects a global network adjustment of multiple regulatory mechanisms.
The mapping and overall sequence similarities of E. salsugineum and Arabidopsis thaliana, together with that of S. parvula provide unique opportunities for tracing evolutionary rearrangements and, perhaps, identifying sources of their differing environmental tolerances.
In addition to the genome reports, there are a number of more other sequencing projects of particular interest in understanding eXtreme adaptations.
Genome wide siRNA expression and the effects of salinity were considered in a separate study of 82 conserved and 17 novel miRNAs.16 Members of 11 conserved miRNA families and 4 novel ones showed “significant responses to salt stress.” Overall, more than 1000 potential targets were predicted with widely varying functions. Additionally, numerous putative stress and phytohormone cis-regulatory elements were identifiable in the promoter regions of salt-responsive miRNA precursors. The sequences of the miRNAs and their targets are available only via the supporting information on the publisher’s website.
In constructing and annotating a reference transcriptome and an associated microarray platform, Lee et al. 17 also addressed the miRNA population, identifying 384 putative miRNAs and their targets. Whether the correct estimate is 82, 141, 384 or something else, the role of miRNAs in stress responses will be an important area for future research.
The availability of multiple genotypes, two sequenced genomes, and ecological and physiological differences between genotypes portends many avenues for future mechanistic studies.