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

Abstract

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.

Securing food for a growing human population is a grand challenge to humanity, which will require solutions stemming from basic sciences (United Nations Framework Convention on Climate Change, 2016; Food and Agriculture Organization of the United Nations, 2017). Saline soils are one of the most serious environmental factors limiting crop productivity. Although assessments vary, it is estimated that up to 10% of the Earth’s surface is affected by salt and approximately 50% of irrigated land (Flowers and Yeo, 1995; Polle and Chen, 2015). In some areas, 67% of agricultural land can be potentially affected by transient salinity (Rengasamy, 2006). The economic cost of crop losses due to salinity, estimated at about US$27 billion annually, in addition to the serious impact on global food security due to increasing salinization of soils (Qadir et al., 2014), is driving the need to generate crops with improved tolerance to salt stress and to develop halophyte-based agriculture (Bressan et al., 2011; Ventura et al., 2015).

Much of our understanding of plant responses to salt stress has been gleaned from a limited number of model plants or economically valuable crop species. In particular, investigations using Arabidopsis (Arabidopsis thaliana) have provided a wealth of information on the physiological and molecular mechanisms of salt tolerance due to its many qualities as a model organism and unmatched genetic and genomic resources. However, Arabidopsis is by no means a stress-tolerant species and is unlikely to possess many of the salt tolerance determinants that are naturally selected and exist in halophytes. These plants grow and reproduce in salt concentrations that would kill 99% of other plant species (Flowers and Colmer, 2008), and studying how halophytes thrive in such saline environments could act as a guide to improving the performance of nonhalophytic crop species (Shabala, 2013).

Characterization of the physiological mechanisms of salt tolerance in halophytes has advanced over the decades, although much is still unknown (Flowers et al., 1977, 2015; Flowers and Colmer, 2008; Munns and Tester, 2008; Cheeseman, 2013). However, elucidation of the molecular mechanisms and adaptive evolutionary signatures of halophytism has lagged behind physiological characterization due to the absence of a model halophyte with all the attendant molecular and genetic tools. Thus, in 2001, the interest of the plant stress community was piqued by mention of an Arabidopsis relative, commonly referred to as salt cress, from the coast of the Chinese Shandong province that is tolerant to salt stress (Bressan et al., 2001; Zhu, 2001). Furthermore, reports showed that salt cress possessed many of the attributes of a model plant that are also associated with Arabidopsis: small size, short life cycle, self-pollination, copious seed production, relatively small genome (approximately twice that of Arabidopsis), and transformation feasibility (Bressan et al., 2001; Amtmann, 2009). The plant was referred to in the literature by the name Thellungiella halophila but later determined to be the species Thellungiella salsuginea, now named Eutrema salsugineum (Al-Shehbaz and Warwick, 2005; Koch and German, 2013).

Realizing the huge potential for comparative analysis of stress tolerance mechanisms between closely related salt-sensitive and halophytic species, a workshop was held in 2004 to promote E. salsugineum as an Arabidopsis relative model system (Amtmann et al., 2005). Since that pioneering meeting, over 125 articles have been published on E. salsugineum and the related (and even more salt-tolerant [Orsini et al., 2010]) Schrenkiella parvula (formally Thellungiella parvula and Eutrema parvulum). Furthermore, various genetic resources have been generated, including chromosome-level genome assemblies, natural accession collections, full-length cDNA and EST collections, transformation protocols, a plant transformation-competent large-insert DNA library, Arabidopsis lines expressing an E. salsugineum cDNA library, nuclear and organelle genome sequences, microRNA sequences, cDNA microarrays, RNA sequencing data sets (Wang et al., 2004, 2010, 2016, 2018a, 2018b; Wong et al., 2005; Du et al., 2008; Taji et al., 2008; Zhang et al., 2008, 2013; Amtmann, 2009; Dassanayake et al., 2011a; Oh et al., 2012; Wu et al., 2012; Bartels and Dinakar, 2013; Champigny et al., 2013; Lee et al., 2013; Yang et al., 2013; Batelli et al., 2014; Fukami-Kobayashi et al., 2014; Guo et al., 2016; He et al., 2016; Yin et al., 2018), and dedicated Web resources (http://extremeplants.org/). Given their increasing importance as model systems (Box 1), E. salsugineum and S. parvula were ranked by the journal Cell as one of the next top models (Zhu et al., 2015).

The aim of this review, therefore, is to survey what progress has been made in understanding halophytism using comparative physiological and molecular analyses of Arabidopsis and E. salsugineum and/or S. parvula. In general, work that has only analyzed the halophytes alone is not covered, as it is difficult to draw conclusions regarding adaptation to salt stress without reference to a salt-sensitive relative. Similarly, we do not cite reports on the salt tolerance of transgenic Arabidopsis overexpressing E. salsugineum genes, as these articles rarely, if ever, examine whether overexpressing these genes has a different effect from overexpressing the Arabidopsis orthologs.

ABIOTIC STRESS TOLERANCE OF E. SALSUGINEUM AND S. PARVULA

E. salsugineum or S. parvula can be synchronously grown with Arabidopsis for comparative analysis in controlled environments (Fig. 1). On the one hand, E. salsugineum is similar to Arabidopsis in that it possesses a rosette leaf ontology during vegetative growth and it bolts during reproductive growth. However, E. salsugineum morphology can differ greatly in its natural habitat, displaying a complete absence of rosette leaves and developing only cauline leaves on multiple stems (Guevara et al., 2012; Fig. 1D). E. salsugineum also has a different developmental program from Arabidopsis; for instance, one of the principal ecotypes under study (Shandong ecotype) requires vernalization to accelerate flowering, although the plant will flower without vernalization after approximately 10 to 12 months (Inan et al., 2004). It also exhibits seed set and germination over an extended period (Inan et al., 2004). S. parvula shows similar growth rates to Arabidopsis but has an elongated stem with alternate leaves and displays indeterminate growth during vegetative and reproductive phases.

Figure 1.

Growth of Arabidopsis, S. parvula, and E. salsugineum on increasing levels of soil NaCl. A, Seven-day-old Arabidopsis plants were treated with various concentrations of NaCl incrementally until final salt concentrations (0, 100, 250, and 500 mm) were reached 1 week later. Plants were imaged after another 1 week. B, E. salsugineum plants treated in the same manner as in A. C, Forty-five-day-old S. parvula plants were treated with various concentrations of NaCl (0, 50, 100, 400, and 600 mm) for 21 d and imaged on day 70. S. parvula plants were able to complete their life cycle and yield seeds even at extreme salt levels. D, E. salsugineum in the salt-crust soils of the Yukon, Canada. Note the presence of cauline leaves while rosette leaves are absent. (Photograph by Jeff Dedrick.)

At the tissue level, E. salsugineum possesses several features that could contribute to its salt tolerance, such as a second layer of palisade mesophyll cells, a higher stomatal density, and differences in leaf epicuticular wax and lipid concentration/composition, all of which could affect water loss under saline conditions (Teusink et al., 2002; Inan et al., 2004; Xu et al., 2014). S. parvula has a higher stomatal density as well as a higher epicuticular wax content compared with Arabidopsis (Teusink et al., 2002; Orsini et al., 2010). Similar to other halophytes, E. salsugineum roots develop a second layer of endodermis and an additional cortex layer that could restrict ion flow from roots to shoots (Flowers et al., 1986; Inan et al., 2004).

E. salsugineum is more tolerant than Arabidopsis to a number of abiotic stresses, including salt stress (Inan et al., 2004), low-nitrogen stress (Kant et al., 2008), phosphate deprivation (Velasco et al., 2016), high boron levels (Lamdan et al., 2012), and heat stress (Higashi et al., 2013). E. salsugineum also exhibits tolerance to chilling and freezing. However, in terms of comparison with Arabidopsis, the relative tolerance of E. salsugineum depends upon the accessions employed, whether plants are preacclimated, and whether the treatment is short or long term (Lee et al., 2012; Khanal et al., 2015). No firm evidence exists for the increased drought tolerance of E. salsugineum compared with Arabidopsis, although natural variation exists in drought tolerance between E. salsugineum accessions (MacLeod et al., 2015). However, E. salsugineum leaves do have constitutively lower water content than Arabidopsis and are able to lose more water in response to osmotic stress, suggesting that E. salsugineum can tolerate dehydration to a greater extent than Arabidopsis (Lugan et al., 2010). Accordingly, E. salsugineum also exhibits greater tolerance to polyethylene glycol-induced osmotic stress than Arabidopsis (Yu and Li, 2014). In addition to high-Na⁺ stress, S. parvula is adapted to multiple-ion stresses, including tolerance to K⁺, Li⁺, and Mg²⁺ at levels toxic to most plants, including E. salsugineum and Arabidopsis (Oh et al., 2014).

The great majority of studies have focused upon E. salsugineum responses to salt stress. Unlike Arabidopsis, this plant is able to survive and complete its life cycle in extreme salt concentrations up to 500 mm NaCl (Inan et al., 2004; Taji et al., 2004; Vera-Estrella et al., 2005; Kant et al., 2006; Ghars et al., 2008; Lugan et al., 2010; Guo et al., 2012). These reports have compared fresh and dry weight reductions as a marker of stress tolerance and have shown that, while salt stress-mediated growth reduction does take place in E. salsugineum, it occurs at higher salt concentrations than for Arabidopsis. However, a more detailed comparative phenomics study unmasked a reduction in E. salsugineum total rosette area by salt concentrations as mild as 50 mm NaCl (Kazachkova et al., 2013). This is likely an adaptive response, in agreement with findings that plants actively reduce their growth in response to stress independently of photosynthesis (Skirycz and Inzé, 2010; Skirycz et al., 2010). The more recently introduced halophytic model, S. parvula, shows a greater capacity to withstand higher NaCl levels than E. salsugineum without compromising growth (Orsini et al., 2010; Oh et al., 2014).

CONTROL OF Na⁺-K⁺ ION TRANSPORT

It has long been dogma that all plants must prevent the accumulation of toxic levels of salts in the cytosol, although what exactly constitutes toxic cytosolic levels of Na⁺ ions is still unclear (Cheeseman, 2013; Flowers et al., 2015). Nevertheless, halophytes have evolved more efficient mechanisms to control cytosolic Na⁺ levels by a combination of exclusion, sequestration, osmotic adjustment, and redistribution (Flowers and Colmer, 2015).

Several studies have shown that, under control conditions, E. salsugineum and S. parvula shoots possess higher levels of Na⁺ than Arabidopsis (Inan et al., 2004; Kant et al., 2006; Wang et al., 2006; Orsini et al., 2010; Oh et al., 2014). Consistent with higher Na⁺ accumulation, E. salsugineum exhibits a constitutively more negative leaf osmotic potential than Arabidopsis in the absence of external NaCl (Inan et al., 2004). Under increasing levels of external NaCl, both E. salsugineum and S. parvula accumulate Na⁺ to a far lower extent than Arabidopsis while maintaining shoot K⁺ levels, suggesting that the halophytes can more tightly control Na⁺ uptake (Inan et al., 2004; Kant et al., 2006; Orsini et al., 2010; Oh et al., 2014). This notion has been confirmed by radiotracer studies demonstrating that the steady-state unidirectional Na⁺ influx into the roots is approximately 50% lower in E. salsugineum than in Arabidopsis (Wang et al., 2006). Consistent with the radiotracer data, electrophysiology studies have revealed a significantly smaller Na⁺ inward current in E. salsugineum than in Arabidopsis, due to the higher selectivity for K⁺ over Na⁺ of a putative E. salsugineum voltage-independent channel (Volkov and Amtmann, 2006). In fact, a general feature of E. salsugineum stress tolerance appears to be its ability to tightly control the accumulation of various ions, including Na⁺, K⁺, B(OH)³, PO₄³⁻, and NO₃⁻ (Inan et al., 2004; Kant et al., 2006, 2008; Lamdan et al., 2012; Velasco et al., 2016).

Several transporters have been studied that could contribute to E. salsugineum salt tolerance. In Arabidopsis, the plasma membrane Na⁺/H⁺ antiporter, SALT OVERLY SENSITIVE1 (SOS1), functions in the exclusion of Na⁺ ions from root cells as well as controlling the loading and retrieval of Na⁺ into and from the xylem stream (Shi et al., 2002). An E. salsugineum ortholog of AtSOS1 is essential for E. salsugineum salt tolerance, and RNA interference-based interference of EsSOS1 expression essentially converts E. salsugineum from a halophyte into a salt-sensitive plant (Oh et al., 2007, 2009a, 2009b). Moreover, when expressed in a salt-sensitive yeast strain, EsSOS1 confers greater salt tolerance than SpSOS1 and AtSOS1 (Jarvis et al., 2014). SOS1 appears to have evolved under positive selection in E. salsugineum, with a single amino acid change in the putative autoinhibitory domain necessary but not sufficient for EsSOS1 to confer enhanced salt tolerance. The Na⁺/K⁺ HIGH-AFFINITY K⁺ TRANSPORTERs (EsHKT1 and SpHKT1) also are essential for E. salsugineum and S. parvula salt adaptation (Ali et al., 2012, 2018; Oh et al., 2014). For instance, RNA interference knockdown of EsHKT1;2 leads to increased hypersensitivity to NaCl, greater accumulation of Na⁺ in the shoots, and reduced K⁺ accumulation (Ali et al., 2012, 2013). Conversely, overexpression of SpHKT1;2 in Arabidopsis causes enhanced tolerance to NaCl, decreased Na⁺ accumulation, and increased K⁺ content (Ali et al., 2018). Thus, EsHKT1;2 and SpHKT1;2 have a vital role in Na⁺/K⁺ balance and an essential function in salt tolerance of the halophytes. Moreover, as detailed below (see “E. salsugineum and S. parvula Genomes: A Window into the Genetic Basis of Adaptation to Extreme Environments”), genomic sequence and expression analyses of the HKT gene family in E. salsugineum and S. parvula reveal specific features of adaptation to saline environments.

An increase in tonoplast Na⁺/H⁺ exchange has been observed in E. salsugineum plants under salt stress (Vera-Estrella et al., 2005), suggesting that compartmentalization of Na⁺is also an important feature of E. salsugineum salt tolerance. The Na⁺/K⁺ (NHX1) transporter was identified originally as an Arabidopsis tonoplast Na⁺/H⁺ antiporter (Apse et al., 1999), although it has been suggested that it functions mainly as a K⁺/H⁺ transporter (Jiang et al., 2010; Leidi et al., 2010). An ortholog of AtNHX1 was isolated from E. salsugineum, and loss-of-function transgenic plants exhibited greater sensitivity to salt and osmotic stress than wild-type plants (Wu et al., 2009). Consistent with these results, it also was demonstrated that Na⁺ ions are compartmentalized primarily within the vacuole in E. salsugineum (Wang et al., 2013). However, neither of these reports provided evidence regarding whether EsNHX1 improved salt tolerance compared with AtNHX1 or how much vacuolar Na⁺ was accumulated relative to Arabidopsis.

HALOPHYTIC PHOTOSYNTHETIC PERFORMANCE AND PROTECTIVE ANTIOXIDANT SYSTEMS

In general, salt stress leads to reduced photosynthetic performance due to various factors, including decreased CO₂ diffusionbecause of diminished stomatal aperture, reduced mesophyll CO₂ transport, altered leaf photochemistry and carbon metabolism, and down-regulation of photosynthetic gene expression (Chaves et al., 2009; Bartels and Dinakar, 2013). A detailed comparison of Arabidopsis and E. salsugineum (Shandong ecotype) photosynthetic responses to salt provided insight into the halophyte’s strategy for maintaining photosynthetic performance under saline conditions (Stepien and Johnson, 2009). This study showed that, in Arabidopsis, salt stress causes a reduction in stomatal conductance, net CO₂ assimilation, PSII electron transport, greater cyclic electron flow around PSI, and increased nonphotochemical quenching (NPQ). Thus, Arabidopsis behaves in a similar manner to other species under stress; namely, a stress-mediated decrease in carbon fixation leads to the down-regulation of linear electron transport, reflecting a reduced need for electrons and the need to minimize the generation of damaging reactive oxygen species (ROS; Golding and Johnson, 2003). Simultaneously, there is a rise in NPQ to dissipate excess energy, thereby protecting PSII. This rise in NPQ is supported by a ∆pH generated by cyclic electron transport. On the other hand, E. salsugineum behaves quite differently from Arabidopsis. Even at extreme salt levels, gas exchange is inhibited only marginally, while no increase in cyclic electron transport or NPQ is observed. There is, however, a large rise in PSII electron flow, and at least 30% of this flow can be accounted for by increased levels of a plastid terminal oxidase (PTOX) compared with Arabidopsis under both control and salt stress conditions (Stepien and Johnson, 2009; Wiciarz et al., 2015). The PTOX could act as an alternative electron sink by transferring electrons from plastoquinone to molecular oxygen without generating ROS.

Limitation of Calvin cycle reactions and the consequent leakage of electrons from the photosynthetic electron transport chain are associated with ROS generation leading to cellular damage (Miller et al., 2010). Several reports suggest that E. salsugineum and S. parvula have highly active antioxidant systems. For instance, Uzilday et al. (2015) showed that a number of enzymes involved in ROS scavenging are active in S. parvula under nonsaline conditions, with only a slightly higher activity under a range of soil NaCl concentrations. When activities are measured specifically in chloroplasts, however, they increase in a NaCl dose-dependent manner. Nevertheless, since there was no comparison with enzyme activities in Arabidopsis, it is difficult to conclude anything about ROS-scavenging mechanisms that are unique to the halophyte. Where such comparative analyses have been performed, antioxidant metabolites such as ascorbate, dehydroascorbate, and caffeic acid are either constitutively higher or accumulate to a greater extent in soil-grown E. salsugineum compared with Arabidopsis (Kazachkova et al., 2013; Eshel et al., 2017). Indeed, the avoidance of singlet oxygen production at PSII has been attributed to induction of the ascorbate-glutathione cycle in E. salsugineum (Wiciarz et al., 2018). Similarly, higher levels of thioredoxin are maintained in salt-stressed E. salsugineum, and this finding is correlated with reduced lipid peroxidation (M’rah et al., 2007). Accordingly, E. salsugineum is more tolerant than Arabidopsis to ROS-inducing methyl viologen (Taji et al., 2004). Higher expression of E. salsugineum genes encoding components of the antioxidant machinery under both control and salt stress conditions might be responsible for a more active and efficient capacity for coping with ROS (Taji et al., 2004; Gong et al., 2005).

Induction of plant defenses against various stresses can be due to fast and transient production of ROS, such as H₂O₂, which act as a stress signal (Apel and Hirt, 2004). A similar amount of H₂O₂ production is induced in Arabidopsis and E. salsugineum leaves a few hours after a 400 mm NaCl shock treatment, whereas higher induction of H₂O₂ is observed in E. salsugineum roots (Ellouzi et al., 2014). However, a more detailed analysis using plants adapted to milder saline conditions (150 mm for Arabidopsis and 300 mm for E. salsugineum), employing a variety of complementary methods, demonstrated that both isolated thylakoid membranes and chloroplasts from intact E. salsugineum leaf tissue produce more H₂O₂ in both control and saline conditions compared with Arabidopsis (Pilarska et al., 2016). Furthermore, under control conditions, a lower amount of damaging superoxide is generated in E. salsugineum than in Arabidopsis, which is correlated with reduced lipid peroxidation in isolated thylakoids and intact leaves of the halophyte. These findings led the authors to speculate that enhanced leakage of H₂O₂ from E. salsugineum plastids preadapts the halophyte to salt stress by maintaining an up-regulated antioxidant system while preventing ROS damage. This hypothesis is further supported by the following observations: (1) the reduced activity of H₂O₂-detoxifying enzymes, such as ascorbate peroxidase (APX) in E. salsugineum and similar activities of catalase in E. salsugineum and Arabidopsis (Pilarska et al., 2016); and (2) the ability of E. salsugineum to sustain a highly reduced plastoquinone pool (Wiciarz et al., 2015, 2018). This could facilitate the persistence of the cellular H₂O₂ signal (Bose et al., 2014). On the other hand, the chloroplasts of E. salsugineum are better protected from ROS damage: thylakoid membranes from the halophyte display a higher abundance of tAPX and Fe superoxide dismutase, while the stroma contains greater levels of peroxiredoxins compared with Arabidopsis. Taken together with other reports suggesting the increased abundance/activity of chloroplast redox components and Calvin-Benson cycle proteins in E. salsugineum (Gong et al., 2005; M’rah et al., 2007; Stepien and Johnson, 2009; Chang et al., 2015; Wiciarz et al., 2015), it is suggested that specific ROS signaling signatures and fine-tuning of specific antioxidants could be a hallmark of stress anticipation in the halophyte.

SALT-MEDIATED TRANSCRIPTOME RESPONSES AND METABOLIC REPROGRAMMING

Metabolism

Several groups have shown that salt-mediated metabolic reprogramming of E. salsugineum is clearly distinct from that of Arabidopsis (Gong et al., 2005; Arbona et al., 2010; Lugan et al., 2010; Pedras and Zheng, 2010; Guevara et al., 2012; Kazachkova et al., 2013; Eshel et al., 2017). In general, the same metabolic pathways are regulated in response to salt stress, but most of the detected metabolites accumulate to higher levels in E. salsugineum under control and salt treatments compared with Arabidopsis. The much lower response to stress observed for E. salsugineum metabolites compared with that of Arabidopsis suggests that the halophyte is primed for stress, a theme that also is observed at the transcriptome level (see below). This idea is emphasized by the finding that over 70% of the detected metabolites that are more abundant in E. salsugineum under control conditions exhibit a salt-mediated increase in Arabidopsis while over 50% of metabolites that are less abundant in E. salsugineum display a salt-mediated decrease in Arabidopsis (Lugan et al., 2010). In other words, the Arabidopsis salt metabolic profile partially resembles that of E. salsugineum under control conditions. Comparing salt stress- and osmotic stress-mediated metabolic reprogramming in Arabidopsis and E. salsugineum also reveals that the expected accumulation of solutes to maintain a negative osmotic potential for water uptake is balanced by a decrease in other solutes such that, overall, no osmotic adjustment takes place in either species (Lugan et al., 2010). The more negative osmotic potential of E. salsugineum compared with Arabidopsis is due to the ability of the halophyte to lose more water. Indeed, inspection of global physiochemical properties suggests that the E. salsugineum metabolome is reconfigured to osmoprotection and tolerance of dehydration.

Several metabolic signatures are maintained independent of growth condition or type of stress in E. salsugineum. For instance, the levels of the tricarboxylic acid cycle intermediates, malate and citrate, are higher in E. salsugineum than in Arabidopsis, whereas fumarate and the osmoprotectants raffinose and galactinol display constitutively low amounts in E. salsugineum (Lugan et al., 2010; Kazachkova et al., 2013; Eshel et al., 2017). The levels of these compounds in E. salsugineum could represent E. salsugineum core stress tolerance mechanisms (Kant et al., 2008; Kazachkova et al., 2013). The functional significance of these signatures is unclear at present. It is tempting to speculate that high malate levels might facilitate the increased production of oxaloacetate to provide carbon skeletons for the increased levels of amino acids observed under control conditions in E. salsugineum. Moreover, the continuous use of oxaloacetate could eventually lead to a deficiency in tricarboxylic acid cycle intermediates, such as the observed reduced accumulation of fumarate. It is interesting that E. salsugineum does not accumulate raffinose or its precursor galactinol in response to salt stress, because raffinose and other members of the raffinose family of oligosaccharides accumulate to high levels under control and stress conditions in other extremophytes (Peters et al., 2007; Farrant et al., 2009; Gechev et al., 2012; Benina et al., 2013). It is unclear why galactinol and raffinose do not accumulate in E. salsugineum, but this might be a distinctive trait similar to that in the desiccation-tolerant spike moss Selaginella lepidophylla (Yobi et al., 2012).

A number of metabolites behave differently according to growth conditions, exemplifying the high degree of metabolic plasticity in E. salsugineum (Kazachkova et al., 2013). The levels of many metabolites (including several known to be involved in stress tolerance) are repressed in E. salsugineum when plants are grown in vitro on nutrient agar plates compared with soil-grown plants. This finding is particularly interesting when considering Pro levels. Several groups have reported increased Pro content in control and salt-stressed E. salsugineum compared with Arabidopsis (Inan et al., 2004; Taji et al., 2004; Kant et al., 2006; Ghars et al., 2008), and it has been suggested that Pro contributes to E. salsugineum salt tolerance as an osmoprotectant and possible ROS scavenger. However, all these studies were performed with soil-grown plants in growth rooms/cabinets or plants grown using hydroponics. Yet, on the saline soil in its native habitat (Yukon ecotype), E. salsugineum exhibits a negligible Pro content (Guevara et al., 2012), and it was surmised that the low levels of nitrogen present in the Yukon soils might be responsible for the naturally low E. salsugineum Pro content in the field. This hypothesis was supported by demonstrating that, under nitrogen-limiting conditions in growth cabinets using soil-grown plants, Pro no longer accumulates in response to salt stress. Similarly, under in vitro conditions, E. salsugineum (Shandong ecotype) does not display increased Pro levels in response to salt stress (Kazachkova et al., 2013). Although the in vitro nutrient medium in that report did not contain reduced nitrogen levels, it did contain Suc, which may have increased the carbon-nitrogen ratio in a similar manner to reducing nitrogen levels. The important point here is that, regardless of growth condition-dependent metabolic profiles (and growth phenotypes), E. salsugineum still retains its salt tolerance, and Guevara et al. (2012) suggested that such phenotypic and metabolic adaptive plasticity would allow the flexibility required for an extremophyte lifestyle.

Transcriptome

The salt responsiveness of the E. salsugineum and S. parvula transcriptomes largely mirrors the metabolome (Taji et al., 2004; Gong et al., 2005; Oh et al., 2014). Both microarray and proteome studies show that fewer transcripts and proteins respond to salt stress in E. salsugineum than in Arabidopsis (Taji et al., 2004; Gong et al., 2005; Pang et al., 2010; Vera-Estrella et al., 2014). Exemplifying a stress-primed state at stress-neutral growth conditions, many genes known for their induction upon salt stress in stress-sensitive plants are found at high constitutively expressed levels in E. salsugineum. The stress-mediated induction of transcriptional responses in E. salsugineum does occur but at higher salinity levels than are required for Arabidopsis, indicating a difference in sensitivity to salt between the two species (Gong et al., 2005; Amtmann, 2009). However, it is important to note that preadaptation is not a universal halophytic trait. Comparison of metabolic profiles of the halophytic legume Lotus creticus with its salt-sensitive relatives Lotus corniculatus and Lotus tenuis has demonstrated that the salt response is conserved globally among the three species (Sanchez et al., 2011). Another interesting observation from global studies of E. salsugineum transcript responses is that there is very little overlap of gene expression between salinity, drought, and cold conditions, suggesting that E. salsugineum is more specific in its stress response than stress-sensitive species (Wong et al., 2005, 2006).

The comparative global analyses of Arabidopsis and E. salsugineum transcriptome/proteome/metabolome responses suggest that the differential regulation of a basic set of stress tolerance genes is a crucial component of E. salsugineum salt tolerance. This notion is supported by comparative RNA sequencing and targeted quantitative PCR analyses between Arabidopsis and the halophytes. Orthologous genes with higher basal expression in S. parvula compared with Arabidopsis are enriched in ion transport functions, whereas pathogen-related genes display higher expression in Arabidopsis (Oh et al., 2014). For example, SOS1 expression is constitutively higher in E. salsugineum roots under control conditions compared with Arabidopsis and is induced by salt to a greater extent in E. salsugineum shoots (Taji et al., 2004, 2010; Kant et al., 2006; Oh et al., 2010; Dassanayake et al., 2011b). This increased EsSOS1 expression is correlated with a reduced uptake of Na⁺ ions into the xylem in response to salt stress compared with Arabidopsis (Kant et al., 2006). Another example is the down-regulation of an E. salsugineum PROLINE DEHYDROGENASE (PDH) gene encoding a Pro catabolic enzyme. This down-regulation is correlated with increased levels of the osmoprotectant, Pro, under control and salt stress conditions in E. salsugineum shoots compared with Arabidopsis (Kant et al., 2006). No such down-regulation of PDH is observed in E. salsugineum roots. Accordingly, Pro levels are similar in E. salsugineum and Arabidopsis roots. It is also notable that the early phospholipase C and D lipid signaling pathways in E. salsugineum control Pro accumulation under non-salt stress or salt stress conditions in an opposite manner to Arabidopsis (Ghars et al., 2012). However, this control does not appear to be exerted via the transcriptional regulation of genes involved either in Pro biosynthesis or catabolism.

E. SALSUGINEUM AND S. PARVULA GENOMES: A WINDOW INTO THE GENETIC BASIS OF ADAPTATION TO EXTREME ENVIRONMENTS

The phenotypic, physiological, biochemical, molecular, and omics studies described above have provided evidence to account for halophyte stress tolerance. However, the missing tool to link genetic adaptation to stress-tolerant phenotypes has been provided by the genome sequences of E. salsugineum and S. parvula and the powerful comparative analysis of these genomes with each other and with that of Arabidopsis (Oh et al., 2010, 2012, 2014; Dassanayake et al., 2011a; Dittami and Tonon, 2012; Wu et al., 2012; Bressan et al., 2013; Yang et al., 2013). The two extremophyte genomes provide high-quality references (Table 1) because they span large intergenic regions beyond the fragmented short scaffolds and minimum curation released for many draft genomes. The S. parvula genome in particular provides one of the most contiguous gap-free assemblies available among plant genomes (Dassanayake et al., 2011a). Thus, the extremophyte genomes are powerful platforms for comparative genomic analyses with established models such as Arabidopsis, rice (Oryza sativa), and poplar (Populus trichocarpa). The two extremophyte genomic resources are being actively updated. For example, new transcripts were reported for E. salsugineum (Yin et al., 2018), and an updated genome assembly and annotation for S. parvula was released via http://thellungiella.org/data/. The genomic resources also are shared via publicly accessible databases, including NCBI, PLAZA, CoGe, Phytozome, and eXtremeplants (http://extremeplants.org).

Table 1. Comparison of Arabidopsis, S. parvula, and E. salsugineum reference genomes.

NA, Not applicable, when the reference publications did not include the relevant value.

Parameter	Arabidopsis	S. parvula	E. salsugineum
	Arabidopsis Genome Initiative	Dassanayake et al. (2011a)	Wu et al. (2012)	Yang et al. (2013)
Predicted genome size (Mb)	150	160	260	NA
Actual genome size (Mb)	115.4	137.09	233.7	241
Total number of genes	27,655	26,847	28,457	26,531
Haploid chromosome number	5	7	7	7
Total number of assembled scaffolds	NA	7	515a	639
N50 (bp), contigs	NA	5.29 Mb	NA	272 kb
N50 (bp), scaffolds	NA	NA	403,516	8 Mb
Number of scaffolds at least N50	NA	8	119	NA
Transposons (% of total genome size)	13.2	7.5	52	NA
Number of orthologous genesb	19,834	19,280	19,582	NA
Number of orphan genesc	5,019	5,844	6,810	NA
Percentage of functionally unknown genes	36.5	62.8	54.7	NA
Average gene length (bp)	2,374	2,110	2,041	NA
Longest open reading frame (bp)	NA	16,758	NA	NA
Average exon length (bp)	224	230	228	NA
Average intron length (bp)	157	191	200	NA

^aScaffolds anchored to seven chromosomes.

^bGenes were considered orthologs if they shared more than 50% amino acid sequence similarity using BLASTP among the three genomes, e < 0.00001.

^cGenes lacking orthologs in the other two species.

Several features of the halophyte genomes provide hypotheses to explain the genetic basis for plant adaptations to extreme environments. Both the E. salsugineum and S. parvula genomes show extensive macrosynteny with the Arabidopsis genome (Dassanayake et al., 2011a; Wu et al., 2012). However, local microsynteny between the species is disrupted by tandem gene duplications, translocations, deletions, and insertions (Oh et al., 2014). Comparisons between the Arabidopsis and S. parvula genomes show that orthologous pairs that have undergone such genome structural variation in either species exhibit less similarity in the putative promoter regions (upstream of the translation start site) than promoters between collinear orthologs. Accordingly, noncollinear genes between the two species exhibit differential basal expression compared with collinear genes (Oh et al., 2014). Moreover, the genes that show genome structural variation in the S. parvula and E. salsugineum genomes via tandem duplications or translocation-duplications are enriched in abiotic stress-associated functions compared with the orthologs in Arabidopsis (Dassanayake et al., 2011a; Wu et al., 2012; Oh et al., 2014). Most of these genomic structural variations also affect gene copy number variation (CNV), which, for decades, has been proposed as a major driver of adaptation to new environments (Ohno, 1970; DeBolt, 2010). Table 2 lists genes associated with stress responses in Arabidopsis that show CNV unique to S. parvula and E. salsugineum based on a network analysis using synteny information from Brassicaceae genomes (Oh and Dassanayake, 2018).

Table 2. Genes that have experimentally verified functions associated with stress responses in Arabidopsis and that show increased copy number in S. parvula and E. salsugineum.

The selected duplication events were not detected in other Brassicaceae lineage I and II genomes in a multispecies comparison including Arabidopsis, Arabidopsis lyrata, Capsella rubella, and Sisymbrium irio genomes (selected subset from Oh and Dassanayake, 2018).

Arabidopsis Locus	Gene Name	Copy Number (At, Es, Sp)	Experimentally Verified Functions	References
AT2G26430	ARGININE-RICH CYCLIN1 (RCY1)	1, 3, 2	Confers tolerance to LiCl and NaCl	Forment et al. (2002)
AT4G30380	BARWIN-RELATED ENDOGLUCANASE	1, 2, 2	Induced during hypoxia	Yang et al. (2011)
AT1G10570	OVERLY TOLERANT TO SALT2 (OTS2)	1, 2, 2	ots1ots2 double mutants show extreme sensitivity to salt	Conti et al. (2008)
AT4G26080	ABA INSENSITIVE1 (ABI1)	1, 2, 2	Reduced sensitivity to salt and osmotic stress during germination	Krzywińska et al. (2016)
AT5G11260	ELONGATED HYPOCOTYL5 (HY5)	1, 3, 3	Role in cold acclimation	Catalá et al. (2011); An et al. (2017)
AT4G20370	TWIN SISTER OF FT (TSF)	2, 4, 5	Role in drought-escape response	Riboni et al. (2013)
AT1G47510	INOSITOL POLYPHOSPHATE 5-PHOSPHATASE11 (5PTASE11)	1, 2, 2	Role in salt-induced ROS production	Kaye et al. (2011)
AT5G59820	RESPONSIVE TO HIGH LIGHT41 (RHL41)	3, 4, 4	Role in reactive oxygen and abiotic stress signaling	Rizhsky et al. (2004); Davletova et al. (2005)

CNV can affect the transcriptome response by the acquisition of new regulatory elements or modification of existing elements in the duplicated genes, leading to gene subfunctionalization (Duarte et al., 2006; Galimba and Di Stilio, 2015). Notably, a number of ion transporters that show CNV in S. parvula suggest gene subfunctionalization, as manifested by organ-specific expression in roots and shoots of the paralogs compared with the single-copy orthologs in Arabidopsis, which are expressed in both organs (Oh et al., 2014; Fig. 2).

Figure 2.

CNV in the HKT1 gene family indicating subfunctionalization and neofunctionalization following tandem duplication in S. parvula and E. salsugineum. A, The single copy of HKT1 in Arabidopsis (At) is tandemly duplicated in S. parvula (Sp) and E. salsugineum (Es). Collinear genomic regions are shown in At, Sp, and Es, with blue and green histograms indicating normalized RNA coverage (RPM) for shoots and roots under stress-neutral conditions. Orthologous genes are connected with arrows. The tissue-specific expression among halophyte HKT1 paralogs suggests gene subfunctionalization. B, Amino acid alignment and tree structure of the HKT1 proteins. ScTRK1, a yeast K⁺ transporter, was used as an outgroup. The sequences selected are a subset of those published by Ali et al. (2012). The alignment includes Es- and Sp-specific HKT1 copies, with the amino acid substitution from Asn (N) to Asp (D) in the second pore-loop domain (depicted in the red boxes for EsHKT1;2, SpHKT1;2, and ScTRK1 copies, with red letters for the substituted D). This N→D substitution was shown to confer the unique K⁺ transport activity (Ali et al., 2012, 2018; Ali and Yun, 2016) indicating neofunctionalization in the HKT1;2 copies compared with the Arabidopsis ortholog. The maximum likelihood tree based on sequence alignment was generated using MEGA7 with a consensus tree based on 100 bootstrap values (Kumar et al., 2016).

A good example of subfunctionalization is Na⁺/H⁺ EXCHANGER8 (NHX8), encoding a putative Li⁺/H⁺ antiporter that is present as a single-copy gene in Arabidopsis (An et al., 2007). However, S. parvula possesses three tandem duplicates of SpNHX8. Genes encoding SpNHX8;1 and SpNHX8;3 exhibit higher basal expression than AtNHX8 in shoots and roots, while SpNHX8;2, which shows a comparable basal expression to AtNHX8 in roots, is induced by LiCl treatment. Accordingly, S. parvula displays much greater tolerance to high levels of LiCl (Oh et al., 2014), consistent with high Li⁺ levels present in its natural habitat and indicating local edaphic adaptation (Helvaci et al., 2004; Nilhan et al., 2008). Although not yet confirmed with targeted transgenic studies, expansion and subfunctionalization of the SpNHX8 genes may represent a CNV event that underlies the genetic basis for the high Li tolerance in S. parvula.

Certain genes that appear to have strict dosage-dependent functions generally are maintained as a single copy in plant genomes (De Smet et al., 2013). The limitations to their expansion are not always clear, and our knowledge of how these gene networks functionally apply restrictions to CNV is limited. However, genes present as single copies in both Arabidopsis and the two halophytes may have significantly different expression profiles at the transcriptome level (i.e. transcriptional CNV) enabled by the insertion or modifications of regulatory elements. For example, the acquisition of new regulatory elements is well illustrated by the AtSOS1 and VACUOLAR H⁺-PYROPHOSPHATASE (AtAVP1) orthologs in E. salsugineum and S. parvula. SOS1 basal and salt-induced expression is greater in the halophytes than in Arabidopsis. Interestingly, EsSOS1, SpSOS1, and indeed all halophytic SOS1 orthologs examined possess a pyrimidine-rich stretch in the 5′ untranslated region (UTR) as well as a predicted stem-loop structure close to the start codon (Dassanayake et al., 2011b). Although the significance of these observations has yet to be demonstrated, such sequences and structures in 5′ UTRs have been associated with enhanced basal gene expression, mRNA stability, and translation efficiency (An and Meagher, 2010; Grillo et al., 2010; Wever et al., 2010). Similarly, the promoter of EsAVP1 can drive the salt-meditated induction of gene expression, unlike the AtAVP1 promoter (Sun et al., 2010). Deletion analysis of the EsAVP1 promoter revealed a 130-bp region that is sufficient for the salt stress response. Further examination of conserved motifs in this region indicated the presence of novel regulatory elements that may provide a genetic basis for this salt-adapted response.

In contrast to the challenge of maintaining high K⁺ uptake while lowering Na⁺ intake that is faced by E. salsugineum and many other plants, S. parvula must limit the uptake of both ions because soils in its natural habitat contain toxic levels of K⁺ and Na⁺ (Ozfidan-Konakci et al., 2016). Gene duplication followed by neofunctionalization and subfunctionalization of the Na⁺/K⁺-transporter gene HKT1 in E. salsugineum and S. parvula illustrates how the two halophytes differentially express HKT1 copies to adapt to specific challenges unique to their niches (Fig. 2). HKT1, a single-copy gene in Arabidopsis, is tandemly duplicated in E. salsugineum and S. parvula (Dassanayake et al., 2011a; Wu et al., 2012). A single amino acid substitution in the duplicated E. salsugineum paralog shifts its function from being a more Na⁺-specific transporter to a more K⁺-specific transporter (Ali et al., 2012, 2016; Ali and Yun, 2016). Under high-Na⁺ stress, E. salsugineum down-regulates the expression of the Na⁺-transporting HKT1 while up-regulating the expression of the K⁺-transporting HKT1 copy, thereby enabling greater K⁺ uptake to facilitate growth and development. In contrast, the expression of the S. parvula Na⁺-transporting HKT1 ortholog is down-regulated in both shoots and roots, similar to E. salsugineum, but the K⁺-transporting S. parvula HKT1 paralog displays further gene subfunctionalization: its expression is down-regulated in the roots to limit K⁺ uptake in a high-K⁺ soil but up-regulated in the shoots to allow K⁺ uptake into developing tissue (Oh et al., 2014). Accordingly, S. parvula indeed maintains higher K⁺ levels in shoots compared with E. salsugineum and Arabidopsis (Orsini et al., 2010; Oh et al., 2014). The HKT1 gene duplication may reveal only one contributing factor in understanding the complex trait of maintaining a suitable K⁺-Na⁺ balance in different environments, but it illustrates how comparative genomic and transcriptomic analyses coupled with functional genetic studies using not just Arabidopsis but also the halophytes could lead to the discovery of naturally selected, stress-adaptive mechanisms.

SALT INHIBITION OF HALOPHYTE SEED GERMINATION

It has been known for decades that, counterintuitively, seeds of many halophytes show reduced germination on saline growth media (Gul et al., 2013, and refs. therein). However, research into halophyte seed germination has focused mainly on surveying germination rates in a large variety of halophytes. Nothing was understood regarding the physiological and biochemical basis of the salt inhibition of halophyte seed germination, let alone the molecular mechanisms regulating this process.

Freshly harvested E. salsugineum seeds (Hebei ecotype) that have not undergone cold treatment display primary physiological dormancy and require up to several months of after-ripening to achieve dormancy release (Li et al., 2015). However, cold stratification of seeds for a few days leads to rapid germination of the principal research ecotypes (e.g. Shandong; Kazachkova et al., 2016). Similar to other halophytes, E. salsugineum and S. parvula seed germination is reduced by NaCl and inhibited completely above a certain NaCl threshold concentration (Inan et al., 2004; Orsini et al., 2010; Guo et al., 2012; Li et al., 2015; Kazachkova et al., 2016). In contrast, at a NaCl concentration that fully inhibits E. salsugineum and S. parvula seed germination, Arabidopsis seeds are still able to germinate. Kazachkova et al. (2016) performed a detailed examination of the physiological, biochemical, and gene expression changes taking place during salt inhibition of E. salsugineum seed germination and showed that seeds respond to the osmotic component of the salt treatment. The seeds remain viable on the saline medium but seed protein and lipid reserves are not mobilized, and the progression of germination-associated metabolic changes is either reduced or halted between 24 and 48 h after stratification. Furthermore, salt-treated seeds exhibit molecular features associated with dormancy, including a reduced GA₃-abscisic acid ratio, induction of stress tolerance-linked processes, and increased expression of dormancy-related genes such as DELAY OF GERMINATION1 (DOG1), compared with non-salt-treated control seeds. However, the observation that salt-inhibited seeds germinate rapidly on salt if the seed coat is removed or if intact seeds are transferred to nonsaline medium suggests that salt does not cause secondary dormancy. Rather, the seeds enter into a dormancy-like state. The physical constraint to seed germination provided by the seed coat is consistent with the higher expression of DOG1 in salt-treated seeds, because DOG1 is involved in repressing the germination-associated weakening of the micropylar endosperm cap (Graeber et al., 2014). Overall, the entry of germinating E. salsugineum seeds into a dormancy-like state in response to the osmotic component of salinity could allow seeds to remain viable in the soil seed bank until rain or melting snow causes a rise in soil water potential and conditions arise that are more favorable to seedling establishment and survival. E. salsugineum now provides an excellent model system to unravel the molecular mechanisms controlling the salt inhibition of halophytic seed germination, and screening of an ethyl methanesulfonate-derived mutant population to identify genes involved in this process is under way (G. Batelli, M. Dassanayake, and S. Barak, unpublished data).

CONCLUDING REMARKS AND FUTURE PERSPECTIVES

We started this review by asking what has been learned about halophytism by comparing glycophytic Arabidopsis with its halophytic relatives, E. salsugineum, and S. parvula. First, it should be noted that much of the work with these halophytes has confirmed previous findings with other halophytic species. These findings include tighter control of Na⁺ uptake, compartmentalization of Na⁺, the importance of plasma membrane and tonoplast Na⁺ transporters in maintaining ionic balance, synthesis of compatible osmolytes, and activation of ROS signaling and detoxification systems (Fig. 3). On the other hand, salt-sensitive plants also employ similar mechanisms to cope with salt stress, suggesting that differences in salt tolerance between halophytes and salt-sensitive species are related to alterations in the regulation of basic physiological and biochemical processes. This hypothesis has been borne out by the advances made studying E. salsugineum and S. parvula (Fig. 3). (1) The halophytic Arabidopsis relatives appear to exhibit global preadaptation of the transcriptome, proteome, and metabolome to stress and differential expression/accumulation of specific stress tolerance genes, proteins, and metabolites. At the same time, they exhibit phenotypic and metabolic flexibility in the face of changing environmental conditions while maintaining salt tolerance. (2) The maintenance of energy supply under saline conditions is clearly a crucial component of halophytism and is at least partly achieved by a combination of alternative pathways for the flow of excess electrons, such as increased levels of PTOX, and the protection of chloroplasts via an up-regulated plastid antioxidant system. (3) Many of the physiological and molecular salt tolerance mechanisms of E. salsugineum and S. parvula are reflected at the genome level. Here, the power of using Arabidopsis relatives in understanding halophytism, at least in Brassicaceae, really comes into its own. Thus, comparative analysis of E. salsugineum and S. parvula genomes with that of Arabidopsis has shown that tandem duplications, insertions, and translocations in the halophytes have led to the expansion of selected genes and alterations in the expression and/or function of these genes via subfuctionalization and/or neofuctionalization. Many of the altered genes can be identified readily as possessing functions that would be important for adaptation to a stressful environment in general and saline conditions specifically.

Figure 3.

Summary of physiological and molecular adaptations that facilitate a halophytic lifestyle. Only those mechanisms demonstrated so far to exhibit differences in E. salsugineum and S. parvula compared with Arabidopsis are depicted. CNV, copy number variation; EsSOS1, E. salsugineum ortholog of the AtSOS1 Na⁺/H⁺ antiporter; EsHKT1, E. salsugineum ortholog of the AtHKT1 Na⁺/K⁺ co-transporter. The genes encoding these transporters have undergone subfunctionalization, neofunctionalization, or both.

Yet, for all that has been revealed about halophytism by studying E. salsugineum and S. parvula, much regarding salt tolerance that is still unexplained in other halophytes (Cheeseman, 2013; Flowers et al., 2015) remains to be elucidated in the halophytic Arabidopsis relatives (see “Outstanding Questions”). Furthermore, detailed physiological investigations that have been carried out in other halophytes need to be performed with the Arabidopsis relatives. This is particularly true for the more salt-tolerant S. parvula, which lags behind the physiological and molecular studies performed with E. salsugineum. For example, rigorous studies of Na⁺ and Cl⁻ ion concentrations in various cell compartments are required to understand the cellular basis of salt tolerance in E. salsugineum and S. parvula (Flowers et al., 2015). Short-distance ion transport and the various transporters that perform this function (Cheeseman, 2013) are poorly understood, while long-distance ion transport (via xylem loading and its regulation) that could be essential for osmotic adjustment in the shoots (Shabala, 2013) has not been investigated at all in the Arabidopsis relatives. We have already seen how E. salsugineum is able to maintain photosynthetic energy supply, but the subject of energy budgets to fuel salt tolerance processes (e.g. Na⁺ vacuolar sequestration, compatible osmolyte production, metabolome and transcriptome reprogramming, etc.) needs to be addressed. Indeed, the contribution of respiration and mitochondria in general has not yet been researched at all in the halophytic Arabidopsis relatives.

One of the general themes from studies with both E. salsugineum and S. parvula is the differential expression of genes between the halophytes and Arabidopsis, whether it is constitutive or more highly salt-induced/-repressed expression of selected genes or at the global transcriptome level. However, the promoter elements, associated transcription factors, and UTR structures involved in this differential halophytic gene expression are unknown, although we have hints of their importance from work on halophytic SOS1 genes and the EsAVP1 gene.

In addition to the acquisition of new regulatory elements, epigenetic factors (DNA methylation and histone modifications) might also be critical for the expression of stress-related genes (Grafi and Barak, 2015; Kim et al., 2015). There are very few reports that have examined epigenetic modifications in extremophytes, but of those that have, some interesting features have emerged. For instance, white mangrove (Laguncularia racemose) located near a salt marsh exhibits global hypomethylation of DNA compared with the same species living next to a freshwater river (Lira-Medeiros et al., 2010). Zygophyllum spp. inhabiting dry and semidry regions lack dimethylation and trimethylation of histone H3 at Lys-9 and Lys-27 (Granot et al., 2009; Granot and Grafi, 2014). Furthermore, the summer dormancy of this plant (leaflets are shed, leaving thick, wax-covered petioles) is associated with the disappearance of permissive H3K4 methylation, leading to a compact chromatin conformation (Khadka et al., 2018). A recent report showed that E. salsugineum has lost gene body DNA methylation (gbM) from its genome and is missing the CHROMOMETHYLASE3 gene that is required for gbM (Bewick et al., 2016). However, no correlation between the loss of gbM and an effect on gene transcription was found. Yet, gbM is only one type of epigenetic feature. Methylation of the promoter regions, histone modifications both globally and locally, and nucleosome occupancy can affect the expression of genes including those involved in stress responses (Grafi and Barak, 2015). The methylation status of transposon remnants also can affect the expression of stress-related genes via transcriptional read-through (Popova et al., 2013). This could be particularly pertinent because of the importance of transposons in generating S. parvula orphan genes (Oh et al., 2012). Thus, epigenetic factors are a topic ripe for investigation in the halophytic Arabidopsis relatives.

Three other features of the halophyte genomes also merit further investigation. (1) Comparison of the E. salsugineum and S. parvula genomes reveals significant differences in repeat-rich transposable elements, where they contribute up to ∼50% of the sequenced genome in E. salsugineum whereas S. parvula has a more compact genome, with less than 10% of the sequenced genome comprising transposable elements (Dassanayake et al., 2011a; Wu et al., 2012). Indeed, several specific transposable element families are underrepresented in the S. parvula genome (Ito et al., 2013; Gao et al., 2016). A preliminary analysis of the two genomes suggested a higher likelihood of differential expression in genes adjacent to transposable elements (Oh et al., 2014). Transposable elements make up a larger fraction of plant genomes than protein-coding genes, thereby providing much of the genomic diversity even in closely related species, with mounting evidence for their role in genome evolution and adaptations (Hollister et al., 2011; Fedoroff, 2012; Oliver et al., 2013; Bennetzen and Wang, 2014; Joly-Lopez et al., 2016, 2017). Yet, the direct influence of transposable elements in contributing to an extremophyte lifestyle is, at present, unexplored. (2) Nearly one-fourth of the genomic sequences under selection in the Arabidopsis genome are made up of noncoding sequences, and a significant fraction of these noncoding sequences are conserved across lineages in Brassicaceae, including in the E. salsugineum and S. parvula genomes (Haudry et al., 2013). Moreover, a large proportion of the conserved noncoding sequences are adjacent to coding regions, yet their functional significance is unknown. In contrast, there is increasing evidence for the functional and evolutionary significance of noncoding RNA families in the E. salsugineum and S. parvula genomes, but this evidence remains in the realm of in silico predictions (Zhang et al., 2013; Rathore et al., 2016; Wu et al., 2016). (3) While there can be many novel or modified functions in the orthologous genes found in the extremophytes that are not yet described in Arabidopsis, there also is a significant fraction of lineage-specific genes with unknown functions in the extremophyte genomes (Dassanayake et al., 2011a; Wu et al., 2012; Yang et al., 2013). Eleven percent of the annotated, nontransposon putative protein-coding genes from S. parvula show no sequence similarity with Arabidopsis genes, while about 7% of these orphan genes show no similarity with any known plant sequence (Dassanayake et al., 2011a; Oh et al., 2012). These genes could represent a pool of unique stress tolerance determinants.

The research areas discussed above are only a few of the many topics that must be addressed both individually and, ultimately, in a holistic manner so that we can understand how the various physiological, biochemical, and molecular mechanisms are integrated to allow halophytes to flourish in saline environments (Cheeseman, 2013). However, at least two obstacles currently prevent greater progress in researching halophytic Arabidopsis relatives. The first problem relates to the variety of nonecologically relevant growth regimes and salt treatments, ranging from hydroponics to soil and from salt shock at extreme salt levels to milder treatments allowing plants to adapt to the applied treatment. E. salsugineum displays very different phenotypic and metabolic characteristics when grown in the laboratory versus plants found in the field, where concentrations of other ions in the soil are of major importance (Guevara et al., 2012; Kazachkova et al., 2013). It is vital, then, that we grow the halophytic Arabidopsis relatives under growth and treatment regimes that reflect at least a modicum of their natural environment. As pointed out by Cheeseman (2013), salt shock treatments should stop. The level of salt given also needs careful consideration, because the E. salsugineum transcriptome/proteome/metabolome respond to concentrations of salt that are considerably higher than those at which Arabidopsis responds. We also must recognize that the halophytes do not experience just salinity in their natural habitats but rather a combination of abiotic stresses. A few groups have begun such combined stress experiments (Khanal et al., 2015; Shamustakimova et al., 2017), but much more needs to be done in this area.

The second obstacle to progress with halophytic Arabidopsis relatives is the poor availability of functional genomics tools. There are no widely available mutant populations for detailed study of individual genes, transgenic lines for tissue/cell specific expression analysis, or the plethora of other tools and databases available for Arabidopsis (Box 1). The published genome sequences of E. salsugineum and S. parvula are a large step in the right direction, but the development of a rich research platform is vital if we are to truly tap the huge potential of the halophytic Arabidopsis relatives.

Dive Curated Terms

The following phenotypic, genotypic, and functional terms are of significance to the work described in this paper:

• AtAVP1 Gramene: At1g15690

• AtAVP1 Araport: At1g15690

• AtNHX8 Gramene: At1g14660

• AtNHX8 Araport: At1g14660

• AtSOS1 Gramene: At2g01980

• AtSOS1 Araport: At2g01980

• EsHKT1;2 Gramene: Ts6g08730

• EsHKT1;2 Araport: Ts6g08730

• EsSOS1 Gramene: Ts2g17860

• EsSOS1 Araport: Ts2g17860

• PROLINE CHEBI: CHEBI:26271

• SpHKT1;2 Gramene: Sp6g07120

• SpHKT1;2 Araport: Sp6g07120

• SpNHX8;1 Gramene: Sp1g13020

• SpNHX8;1 Araport: Sp1g13020

• SpNHX8;2 Gramene: Sp1g13030

• SpNHX8;2 Araport: Sp1g13030

• SpNHX8;3 Gramene: Sp1g13040

• SpNHX8;3 Araport: Sp1g13040

• SpSOS1 Gramene: Sp2g13410

• SpSOS1 Araport: Sp2g13410

• salt CHEBI: CHEBI:24866

• AtNHX1 Gramene: At5g27150

• AtNHX1 Araport: At5g27150

• AtHKT1 Gramene: At4g10310

• AtHKT1 Araport: At4g10310

• EsHKT1;1 Gramene: Ts6g08650

• EsHKT1;1 Araport: Ts6g08650

• EsHKT1;3 Gramene: Ts6g08740

• EsHKT1;3 Araport: Ts6g08740

• SpHKT1;1 Gramene: Sp6g07110

• SpHKT1;1 Araport: Sp6g07110

• EsNHX1 Gramene: Ts2g25410

• EsNHX1 Araport: Ts2g25410

• AtPDH1 Gramene: At3g30775

• AtPDH1 Araport: At3g30775

• EsPDH1 Gramene: Ts2g23100

• EsPDH1 Araport: Ts2g23100

• AtRCY1 Gramene: At2g26430

• AtRCY1 Araport: At2g26430

• EsRCY1;1 Gramene: Ts1g23650

• EsRCY1;1 Araport: Ts1g23650

• EsRCY1;2 Gramene: Ts4g08710

• EsRCY1;2 Araport: Ts4g08710

• EsRCY1;3 Gramene: Ts7g26530

• EsRCY1;3 Araport: Ts7g26530

• SpRCY1;1 Gramene: Sp1g23300

• SpRCY1;1 Araport: Sp1g23300

• SpRCY1;2 Gramene: Sp4g07260

• SpRCY1;2 Araport: Sp4g07260

• AtBARWIN-RELATED ENDOGLUCANASE Gramene: At4g30380

• AtBARWIN-RELATED ENDOGLUCANASE Araport: At4g30380

• EsBARWIN-RELATED ENDOGLUCANASE;2 Gramene: Ts7g29540

• EsBARWIN-RELATED ENDOGLUCANASE;2 Araport: Ts7g29540

• EsBARWIN-RELATED ENDOGLUCANASE;1 Gramene: Ts7g29530

• EsBARWIN-RELATED ENDOGLUCANASE;1 Araport: Ts7g29530

• SpBARWIN-RELATED ENDOGLUCANASE;1 Gramene: Sp7g28250

• SpBARWIN-RELATED ENDOGLUCANASE;1 Araport: Sp7g28250

• SpBARWIN-RELATED ENDOGLUCANASE;2 Gramene: Sp7g28260

• SpBARWIN-RELATED ENDOGLUCANASE;2 Araport: Sp7g28260

• AtOTS2 Gramene: At1g10570

• AtOTS2 Araport: At1g10570

• EsOTS2 Gramene: Ts1g09190

• EsOTS2 Araport: Ts1g09190

• SpOTS2;1 Gramene: Sp1g09220

• SpOTS2;1 Araport: Sp1g09220

• SpOTS2;2 Gramene: Sp5g18150

• SpOTS2;2 Araport: Sp5g18150

• AtABI1 Gramene: At4g26080

• AtABI1 Araport: At4g26080

• EsABI1;1 Gramene: Ts2g10620

• EsABI1;1 Araport: Ts2g10620

• EsABI1;2 Gramene: Ts7g24900

• EsABI1;2 Araport: Ts7g24900

• SpABI1;1 Gramene: Sp6g00090

• SpABI1;1 Araport: Sp6g00090

• SpABI1'2 Gramene: Sp7g24100

• SpABI1'2 Araport: Sp7g24100

• AtHY5 Gramene: At5g11260

• AtHY5 Araport: At5g11260

• EsHY5;1 Gramene: Ts2g27200

• EsHY5;1 Araport: Ts2g27200

• EsHY5;2 Gramene: Ts6g35270

• EsHY5;2 Araport: Ts6g35270

• EsHY5;3 Gramene: Ts-Un0471_001

• EsHY5;3 Araport: Ts-Un0471_001

• SpHY5;1 Gramene: Sp2g22110

• SpHY5;1 Araport: Sp2g22110

• SpHY5;2 Gramene: Sp4g04910

• SpHY5;2 Araport: Sp4g04910

• SpHY5;3 Gramene: Sp6g32300

• SpHY5;3 Araport: Sp6g32300

• AtTSF Gramene: At4g20370

• AtTSF Araport: At4g20370

• EsTSF;1 Gramene: Ts5g34510

• EsTSF;1 Araport: Ts5g34510

• EsTSF;2 Gramene: Ts7g19290

• EsTSF;2 Araport: Ts7g19290

• SpTSF;1 Gramene: Sp5g32040

• SpTSF;1 Araport: Sp5g32040

• SpTSF;2 Gramene: Sp7g18730

• SpTSF;2 Araport: Sp7g18730

• At5PTASE11 Gramene: At1g47510

• At5PTASE11 Araport: At1g47510

• Es5PTASE11 Gramene: Ts1g35030

• Es5PTASE11 Araport: Ts1g35030

• Sp5PTASE11;1 Gramene: Sp1g34540

• Sp5PTASE11;1 Araport: Sp1g34540

• Sp5PTASE11;2 Gramene: Sp7g05230

• Sp5PTASE11;2 Araport: Sp7g05230

• AtRHL41 Gramene: At5g59820

• AtRHL41 Araport: At5g59820

• EsRHL41 Gramene: Ts6g23920

• EsRHL41 Araport: Ts6g23920

• SpRHL41 Gramene: Sp6g21500

• SpRHL41 Araport: Sp6g21500

• EsAVP1;1 Gramene: Ts1g14070

• EsAVP1;1 Araport: Ts1g14070

• EsAVP1;2 Gramene: Ts5g37710

• EsAVP1;2 Araport: Ts5g37710

• EsAVP1;3 Gramene: Ts6g11420

• EsAVP1;3 Araport: Ts6g11420

• AtDOG1 Gramene: At5g45830

• AtDOG1 Araport: At5g45830

• EsDOG1 Gramene: Ts2g14680

• EsDOG1 Araport: Ts2g14680

• AtCMT1 Gramene: At1g80740

• AtCMT1 Araport: At1g80740

• EsCMT1;1 Gramene: Ts1g09960

• EsCMT1;1 Araport: Ts1g09960

• EsCMT1;2 Gramene: Ts5g29880

• EsCMT1;2 Araport: Ts5g29880