Halophytism: What Have We Learnt From Arabidopsis thaliana Relative Model Systems?

Halophytes are able to thrive in salt concentrations that would kill 99% of other plant species, and identifying their salt-adaptive mechanisms has great potential for improving the tolerance of crop plants to salinized soils. Much research has focused on the physiological basis of halophyte salt tolerance, whereas the elucidation of molecular mechanisms has traditionally lagged behind due to the absence of a model halophyte system. However, over the last decade and a half, two Arabidopsis (Arabidopsis thaliana) relatives, Eutrema salsugineum and Schrenkiella parvula, have been established as transformation-competent models with various genetic resources including high-quality genome assemblies. These models have facilitated powerful comparative analyses with salt-sensitive Arabidopsis to unravel the genetic adaptations that enable a halophytic lifestyle. The aim of this review is to explore what has been learned about halophytism using E. salsugineum and S. parvula We consider evidence from physiological and molecular studies suggesting that differences in salt tolerance between related halophytes and salt-sensitive plants are associated with alterations in the regulation of basic physiological, biochemical, and molecular processes. Furthermore, we discuss how salt tolerance mechanisms of the halophytic models are reflected at the level of their genomes, where evolutionary processes such as subfunctionalization and/or neofunctionalization have altered the expression and/or functions of genes to facilitate adaptation to saline conditions. Lastly, we summarize the many areas of research still to be addressed with E. salsugineum and S. parvula as well as obstacles hindering further progress in understanding halophytism.

The evolution of halophytes, glycophytes and crops, and its implications for food security under saline conditions.

The effective development of salt tolerant crops requires an understanding that the evolution of halophytes, glycophytes and our major grain crops has involved significantly different processes. Halophytes (and other edaphic endemics) generally arose through colonization of habitats in severe disequilibrium by pre-adapted individuals, rather than by gradual adaptation from populations of 'glycophytes'. Glycophytes, by contrast, occur in low sodium ecosystems, where sodium was and is the major limiting nutrient in herbivore diets, suggesting that their evolution reflects the fact that low sodium individuals experienced lower herbivory and had higher fitness. For domestication/evolution of crop plants, the selective pressure was human imposed and involved humans co-opting functions of defense and reproductive security. Unintended consequences of this included loss of tolerance to various stresses and loss of the genetic variability needed to correct that. Understanding, combining and manipulating all three modes of evolution are now critical to the development of salt tolerant crops, particularly those that will offer food security in countries with few economic resources and limited infrastructure. Such efforts will require exploiting the genetic structures of recently evolved halophytes, the genetic variability of model plants, and endemic halophytes and 'minor' crops that already exist.

Insights into salt tolerance from the genome of Thellungiella salsuginea.

Thellungiella salsuginea, a close relative of Arabidopsis, represents an extremophile model for abiotic stress tolerance studies. We present the draft sequence of the T. salsuginea genome, assembled based on ~134-fold coverage to seven chromosomes with a coding capacity of at least 28,457 genes. This genome provides resources and evidence about the nature of defense mechanisms constituting the genetic basis underlying plant abiotic stress tolerance. Comparative genomics and experimental analyses identified genes related to cation transport, abscisic acid signaling, and wax production prominent in T. salsuginea as possible contributors to its success in stressful environments.

Genome structures and halophyte-specific gene expression of the extremophile Thellungiella parvula in comparison with Thellungiella salsuginea (Thellungiella halophila) and Arabidopsis.

The genome of Thellungiella parvula, a halophytic relative of Arabidopsis (Arabidopsis thaliana), is being assembled using Roche-454 sequencing. Analyses of a 10-Mb scaffold revealed synteny with Arabidopsis, with recombination and inversion and an uneven distribution of repeat sequences. T. parvula genome structure and DNA sequences were compared with orthologous regions from Arabidopsis and publicly available bacterial artificial chromosome sequences from Thellungiella salsuginea (previously Thellungiella halophila). The three-way comparison of sequences, from one abiotic stress-sensitive species and two tolerant species, revealed extensive sequence conservation and microcolinearity, but grouping Thellungiella species separately from Arabidopsis. However, the T. parvula segments are distinguished from their T. salsuginea counterparts by a pronounced paucity of repeat sequences, resulting in a 30% shorter DNA segment with essentially the same gene content in T. parvula. Among the genes is SALT OVERLY SENSITIVE1 (SOS1), a sodium/proton antiporter, which represents an essential component of plant salinity stress tolerance. Although the SOS1 coding region is highly conserved among all three species, the promoter regions show conservation only between the two Thellungiella species. Comparative transcript analyses revealed higher levels of basal as well as salt-induced SOS1 expression in both Thellungiella species as compared with Arabidopsis. The Thellungiella species and other halophytes share conserved pyrimidine-rich 5' untranslated region proximal regions of SOS1 that are missing in Arabidopsis. Completion of the genome structure of T. parvula is expected to highlight distinctive genetic elements underlying the extremophile lifestyle of this species.

Comparative transcriptomics for mangrove species: an expanding resource.

We present here the Mangrove Transcriptome Database (MTDB), an integrated, web-based platform providing transcript information from all 28 mangrove species for which information is available. Sequences are annotated, and when possible, GO clustered and assigned to KEGG pathways, making MTDB a valuable resource for approaching mangrove or other extremophile biology from the transcriptomic level. As one example outlining the potential of MTDB, we highlight the analysis of mangrove microRNA (miRNA) precursor sequences, miRNA target sites, and their conservation and divergence compared with model plants. MTDB is available at http://mangrove.illinois.edu .

The integration of activity in saline environments: problems and perspectives.

The successful integration of activity in saline environments requires flexibility of responses at all levels, from genes to life cycles. Because plants are complex systems, there is no 'best' or 'optimal' solution and with respect to salt, glycophytes and halophytes are only the ends of a continuum of responses and possibilities. In this review, I briefly examine seven major aspects of plant function and their responses to salinity including transporters, secondary stresses, carbon acquisition and allocation, water and transpiration, growth and development, reproduction, and cytosolic function and 'integrity'. I conclude that new approaches are needed to move towards understanding either organismal integration or 'salt tolerance', especially cessation of protocols dependent on sudden, often lethal, shock treatments and the embracing of systems level resources. Some of the tools needed to understand the integration of activity and even 'salt stress' are already in hand, such as those for whole-transcriptome analysis. Others, ranging from discovery studies of the nature of the cytosol to expanded tool kits for proteomic, metabolomic and epigenomic studies, still need to be further developed. After resurrecting the distinction between applied stress and the resultant strain and noting that with respect to salinity, the strain is manifest in changes at all -omic levels, I conclude that it should be possible to model and quantify stress responses.

Life at the extreme: lessons from the genome.

Extremophile plants thrive in places where most plant species cannot survive. Recent developments in high-throughput technologies and comparative genomics are shedding light on the evolutionary mechanisms leading to their adaptation.

The genome of the extremophile crucifer Thellungiella parvula.

Thellungiella parvula is related to Arabidopsis thaliana and is endemic to saline, resource-poor habitats, making it a model for the evolution of plant adaptation to extreme environments. Here we present the draft genome for this extremophile species. Exclusively by next generation sequencing, we obtained the de novo assembled genome in 1,496 gap-free contigs, closely approximating the estimated genome size of 140 Mb. We anchored these contigs to seven pseudo chromosomes without the use of maps. We show that short reads can be assembled to a near-complete chromosome level for a eukaryotic species lacking prior genetic information. The sequence identifies a number of tandem duplications that, by the nature of the duplicated genes, suggest a possible basis for T. parvula's extremophile lifestyle. Our results provide essential background for developing genomically influenced testable hypotheses for the evolution of environmental stress tolerance.

Seasonal patterns of leaf H2O2 content: reflections of leaf phenology, or environmental stress?

H2O2 is an ubiquitous compound involved in signalling, metabolic control, stress responses and development. The compatibility of leaf tissue levels with these functions has, however, often been questioned. The objective here is to document H2O2 levels and variability under natural conditions, and their underlying causes. Using the FOX method, bulk H2O2 concentrations were analysed in leaf samples from 18 species of herbs and trees throughout the 2006 growing season. Sampling addressing targeted predictions was emphasised in 2007 and 2008. H2O2 levels varied 100-fold through the year, with a main peak in spring. Two hypotheses were examined: (H1) that H2O2 reflects seasonally variable responses to environmental stresses, and (H2) that it reflects metabolism associated with leaf development. Based on poor or inappropriate correlations between H2O2 and indicators of light, temperature or drought stress, support for H1 was minimal. H2 was supported both by seasonal patterns and by targeted analyses of concentration changes throughout leaf development. This study concludes that bulk tissue H2O2 concentrations are poor indicators of stress, and are generally too high to reflect either signalling or metabolic control networks. Instead, the linkage of H2O2 and leaf phenology appears to reflect the roles of H2O2 in cell expansion, lignification and wall cross-linking.

Hydrogen peroxide concentrations in leaves under natural conditions.

While H2O2 has been implicated in numerous plant environmental responses, normal levels and variabilities are poorly established, and estimates of leaf tissue concentrations span more than three orders of magnitude, even in a single species under similar conditions. Here, leaf tissue H2O2 contents under natural conditions are reported after determining (i) that H2O2 in extracts was stable with time, (ii) that H2O2 added to the extract was recovered quantitatively, and (iii) that the H2O2 calibration curve was unaffected (or quantifiably affected) by the extract. The broad applicability of the protocol and variability in leaf concentrations were demonstrated using tissue collected from several habitats in association with three, more extensive, experiments. The first involved nychthemeral studies of the mangrove, Rhizophora mangle L. Lowest H2O2 levels occurred in the early morning and near sunset, with higher levels both at midday and at night. Second, using five temperate species in Spring, concentrations were compared on a warm, sunny day and a cool, cloudy day. Higher concentrations were found on the warm day for Aesculus glabra Willd., Glechoma hederacea L., Plantago major L., and Viola soraria Willd., while there were no differences in Quercus macrocarpa Michx. Finally, the effects of elevated CO2 and ozone were examined in soybean, Glycine max L. Pioneer 93B15 under Free Air gas Concentration Enrichment (FACE) conditions. Both supplements led to elevated H2O2. Overall, mean leaf, midday, and mid-summer H2O2 concentrations ranged from 0.67 micromol (gFW)(-1) in mangrove to 3.6 micromol (gFW)(-1) in A. glabra Willd. Greatest within-species differences were only 2.5-fold in any of the studies.