Alkali cation exchangers: roles in cellular homeostasis and stress tolerance

Uptake and translocation of cations play essential roles in plant nutrition, signal transduction, growth, and development. Among them, potassium (K+) and sodium (Na+) have been the focus of numerous physiological studies because K+ is an essential macronutrient and the most abundant inorganic cation in plant cells, whereas Na+ toxicity is a principal component of the deleterious effects associated with salinity stress. Although the homeostasis of these two ions was long surmised to be fine tuned and under complex regulation, the myriad of candidate membrane transporters mediating their uptake, intracellular distribution, and long-distance transport is nevertheless perplexing. Recent advances have shown that, in addition to their function in vacuolar accumulation of Na+, proteins of the NHX family are endosomal transporters that also play critical roles in K+ homeostasis, luminal pH control, and vesicle trafficking. The plasma membrane SOS1 protein from Arabidopsis thaliana, a highly specific Na+/H+ exchanger that catalyses Na+ efflux and that regulates its root/shoot distribution, has also revealed surprising interactions with K+ uptake mechanisms by roots. Finally, the function of individual members of the large CHX family remains largely unknown but two CHX isoforms, AtCHX17 and AtCH23, have been shown to affect K+ homeostasis and the control of chloroplast pH, respectively. Recent advances on the understanding of the physiological processes that are governed by these three families of cation exchangers are reviewed and discussed.     

Europe‐wide biogeographical patterns in the diet of an ecologically and epidemiologically important mesopredator,the red fox Vulpes vulpes: a quantitative review

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Population differentiation of the Chagas disease vector Triatoma maculata (Erichson, 1848) from Colombia and Venezuela

The emerging vector of Chagas disease, Triatoma maculata (Hemiptera, Reduviidae), is one of the most widely distributed Triatoma species in northern South America. Despite its increasing relevance as a vector, no consistent picture of the magnitude of genetic and phenetic diversity has yet been developed. Here, several populations of T. maculata from eleven Colombia and Venezuela localities were analyzed based on the morphometry of wings and the mitochondrial NADH dehydrogenase subunit 4 (ND4) gene sequences. Our results showed clear morphometric and genetic differences among Colombian and Venezuelan populations, indicating high intraspecific diversity. Inter‐population divergence is suggested related to East Cordillera in Colombia. Analyses of other populations from Colombia, Venezuela, and Brazil from distinct eco‐geographic regions are still needed to understand its systematics and phylogeography as well as its actual role as a vector of Chagas disease.     

A constitutively active form of a durum wheat Na+/H+ antiporter SOS1 confers high salt tolerance to transgenic Arabidopsis

Key message

Expression of a truncated form of wheat TdSOS1 in Arabidopsis exhibited an improved salt tolerance. This finding provides new hints about this protein that can be considered as a salt tolerance determinant.

Abstract

The SOS signaling pathway has emerged as a key mechanism in preserving the homeostasis of Na⁺ and K⁺ under saline conditions. We have recently identified and functionally characterized, by complementation studies in yeast, the gene encoding the durum wheat plasma membrane Na⁺/H⁺ antiporter (TdSOS1). To extend these functional studies to the whole plant level, we complemented Arabidopsis sos1-1 mutant with wild-type TdSOS1 or with the hyperactive form TdSOS1?972 and compared them to the Arabidopsis AtSOS1 protein. The Arabidopsis sos1-1 mutant is hypersensitive to both Na⁺ and Li⁺ ions. Compared with sos1-1 mutant transformed with the empty binary vector, seeds from TdSOS1 or TdSOS1?972 transgenic plants had better germination under salt stress and more robust seedling growth in agar plates as well as in nutritive solution containing Na⁺ or Li⁺ salts. The root elongation of TdSOS1?972 transgenic lines was higher than that of Arabidopsis sos1-1 mutant transformed with TdSOS1 or with the endogenous AtSOS1 gene. Under salt stress, TdSOS1?972 transgenic lines showed greater water retention capacity and retained low Na⁺ and high K⁺ in their shoots and roots. Our data showed that the hyperactive form TdSOS1?972 conferred a significant ionic stress tolerance to Arabidopsis plants and suggest that selection of hyperactive alleles of the SOS1 transport protein may pave the way for obtaining salt-tolerant crops.     

An Invasive Vector of Zoonotic Disease Sustained by Anthropogenic Resources: The Raccoon Dog in Northern Europe

The raccoon dog (Nyctereutes procyonoides) is an introduced species in Europe with a continually expanding range. Since the species is capable of affecting local ecosystems and is a vector for a number of severe zoonotic diseases, it is important to understand its food habits. Raccoon dog diet was studied in Estonia by examining the contents of 223 stomach samples collected during the coldest period of the year, August to March, in 2010–2012. The most frequently consumed food categories were anthropogenic plants (e.g. cereals, fruits; FO = 56.1%) and carrion (e.g. carcasses of artiodactyls and carnivores; FO = 48.4%). Carrion was also the only food category that was consumed significantly more frequently by raccoon dogs exhibiting symptoms of sarcoptic mange than by uninfected animals. Small mammals, which represent intermediate hosts for the zoonotic tapeworm Echinococcus multilocularis, were more commonly recorded in samples also containing anthropogenic plants than expected by chance. Comparison of raccoon dog and red fox (Vulpes vulpes) diet in Estonia revealed higher overlap than found elsewhere in Europe, with ‘carrion’ and ‘anthropogenic plants’ making up the bulk of both species’ diet; however, raccoon dogs were more omnivorous than red foxes. Our results suggest that while the use of most food categories reflects the phenology of natural food sources, ‘anthropogenic plants’ and ‘carrion’ provide an essential resource for raccoon dogs during the coldest period of the year, with the latter resource especially important for individuals infected with sarcoptic mange. Since both of these food categories and small mammals are often found at supplementary feeding sites for wild boar (Sus scrofa), this game management practice may facilitate high densities of mesocarnivores and promote the spread of some severe zoonotic diseases, including alveolar echinococcosis, trichinellosis, rabies and sarcoptic mange.     

The CCL3 family of chemokines and innate immunity cooperate in vivo in the eradication of an established lymphoma xenograft by rituximab

The therapeutic mAb rituximab induced the expression of the CCL3 and CCL4 chemokines in the human lymphoma line BJAB following binding to the CD20 Ag. Induction of CCL3/4 in vitro was specific, was observed in several cell lines and freshly isolated lymphoma samples and also took place at the protein level in vitro and in vivo. To investigate the role of these beta-chemokines in the mechanism of action of rituximab, we synthesized a N-terminally truncated CCL3 molecule CCL3(11-70), which had antagonist activity on chemotaxis mediated by either CCL3 or BJAB supernatant. We also set up an established s.c. BJAB tumor model in athymic mice. Rituximab, given weekly after tumors had reached 250 mm2, led to complete disappearance of the lymphoma within 2-3 wk. Treatment of mice with cobra venom factor showed that complement was required for rituximab therapeutic activity. Treatment of BJAB tumor bearing mice every 2 days with the CCL3(11-70) antagonist, starting 1 wk before rituximab treatment, had no effect on tumor growth by itself, but completely inhibited the therapeutic activity of the Ab. To determine whether CCL3 acts through recruitment/activation of immune cells, we specifically depleted NK cells, polymorphonuclear cells, and macrophages using mAbs, clodronate treatment, or Rag2-/-cgamma-/- mice. The data demonstrated that these different cell populations are involved in BJAB tumor eradication. We propose that rituximab rapidly activates complement and induces beta-chemokines in vivo, which in turn activate the innate immunity network required for efficient eradication of the bulky BJAB tumor.     

Loss of Halophytism by Interference with SOS1 Expression

The contribution of SOS1 (for Salt Overly Sensitive 1), encoding a sodium/proton antiporter, to plant salinity tolerance was analyzed in wild-type and RNA interference (RNAi) lines of the halophytic Arabidopsis (Arabidopsis thaliana)-relative Thellungiella salsuginea. Under all conditions, SOS1 mRNA abundance was higher in Thellungiella than in Arabidopsis. Ectopic expression of the Thellungiella homolog ThSOS1 suppressed the salt-sensitive phenotype of a Saccharomyces cerevisiae strain lacking sodium ion (Na⁺) efflux transporters and increased salt tolerance of wild-type Arabidopsis. thsos1-RNAi lines of Thellungiella were highly salt sensitive. A representative line, thsos1-4, showed faster Na⁺ accumulation, more severe water loss in shoots under salt stress, and slower removal of Na⁺ from the root after removal of stress compared with the wild type. thsos1-4 showed drastically higher sodium-specific fluorescence visualized by CoroNa-Green, a sodium-specific fluorophore, than the wild type, inhibition of endocytosis in root tip cells, and cell death in the adjacent elongation zone. After prolonged stress, Na⁺ accumulated inside the pericycle in thsos1-4, while sodium was confined in vacuoles of epidermis and cortex cells in the wild type. RNAi-based interference of SOS1 caused cell death in the root elongation zone, accompanied by fragmentation of vacuoles, inhibition of endocytosis, and apoplastic sodium influx into the stele and hence the shoot. Reduction in SOS1 expression changed Thellungiella that normally can grow in seawater-strength sodium chloride solutions into a plant as sensitive to Na⁺ as Arabidopsis.Accompanying the production and accumulation of osmolytes and other protective molecules, an important aspect of plant responses leading to salt stress tolerance is the regulation of uptake, reexport, and control over the distribution of sodium ions (Na⁺; Hasegawa et al., 2000; Tester and Davenport, 2003). Na⁺ appear to enter the root by several pathways (Essah et al., 2003; Pardo et al., 2006), although the nature of participating genes and their interaction in pathways require further investigation. Once Na⁺ has entered the root endodermis, a tissue that represents a barrier to ions (Peng et al., 2004), it is generally assumed that the ion enters the xylem following the movement of water to aerial parts of the plant. Despite substantial efflux of Na⁺ across the plasma membrane of root cells, the net flux of Na⁺ is unidirectional from soil to roots and then to the shoot, except for possible recirculation via the phloem (Tester and Davenport, 2003). In a range of species, the severity of damaging symptoms is positively correlated with the content of Na⁺ reaching photosynthetic tissues (Davenport et al., 2005; Ren et al., 2005; Munns et al., 2006). However, halophytic species can accumulate very high amounts of Na⁺ in vacuoles, such that Na⁺ may account for most of the total cellular osmotic potential (Tester and Davenport, 2003), and the presence of Na⁺ accelerates growth in euhalophytes to some degree (Adams et al., 1998). Emerging as the major advantage of halophytes appears to be their exceptional control over Na⁺ influx combined with export mechanisms, the ability to coordinate its distribution to various tissues, and efficient sequestration of Na⁺ into vacuoles. These characteristics are of particular advantage when plants are subjected to a sudden increase of Na⁺ salts in their environment (Hasegawa et al., 2000), whereas gradual increases in Na⁺ may be tolerated even by plants that are not halophytic in nature.Na⁺-ATPases, major Na⁺ export systems in organisms such as fungi and the moss Physcomitrella patens, have not been found in higher plants (Lunde et al., 2007). In Arabidopsis (Arabidopsis thaliana), transporters of monovalent (alkali) cations, such as HKT1 (Berthomieu et al., 2003; Rus et al., 2004), members of the NHX family (Yamaguchi et al., 2005; Pardo et al., 2006), and SOS1 (for Salt Overly Sensitive 1; Shi et al., 2000, 2002, 2003), have been shown to play roles in the movement and distribution of Na⁺ ions. Studies have shown the involvement of nonselective ion channels with roles in the transport of Na⁺ ions, but the genes encoding such function(s) have not been identified (Demidchik and Maathuis, 2007). SOS1, whose deletion resulted in a strong salt-sensitivity phenotype in Arabidopsis, encodes a plasma membrane Na⁺/H⁺ antiporter involved in removing Na⁺ ions from cells (Shi et al., 2000). This efflux strategy, which may be sufficient for the survival of unicellular organisms, must be accompanied by other means of Na⁺ confinement to avoid carryover of Na⁺ between cells in futile cycles. Hence, the physiological role of a plasma membrane Na⁺/H⁺ antiporter must be embedded in the context of tissue, organ, and whole plant distribution of ions and their transporters. A recent discovery on cell layer-specific differential responses to the salt stress of root cells supported this notion (Dinneny et al., 2008).In Arabidopsis, the SOS1 gene is most strongly expressed in the epidermis of the root tip region and in cells adjacent to vascular tissues (Shi et al., 2002). Based on the salt concentration in shoot, root, and xylem sap of wild-type Arabidopsis and its sos1 knockout mutants, the SOS1 antiporter is assumed to function in Na⁺ export under severe salt stress conditions (Shi et al., 2002). However, detailed knowledge about how a Na⁺ excluder achieves salt tolerance in a multicellular eukaryote is still missing. Significantly also, even though SOS1 has been an intensely studied component of the ion homeostasis mechanism, its involvement in the exceptional salt tolerance of halophytes is not known.Thellungiella salsuginea (salt cress), which had before been called T. halophila by us, is a close relative of Arabidopsis, which has become a model to study the genetic basis of this plant''s extreme tolerance to a variety of abiotic stress factors, including salinity (Inan et al., 2004; Gong et al., 2005; Vera-Estrella et al., 2005; Volkov and Amtmann, 2006; Amtmann, 2009). Thellungiella lacks specialized morphological structures, such as salt glands or large sodium storage cells found in other halophytes, making it a useful model for studying stress tolerance mechanisms that could be applicable to further understanding or to embark on engineering of conventional crops (Inan et al., 2004). Recently, it has been reported that Thellungiella had lower net Na⁺ uptake compared with Arabidopsis. The unidirectional influx of Na⁺ ions to roots appeared to be more restricted and/or tightly controlled in Thellungiella than in Arabidopsis. To compensate for greater influx, Arabidopsis roots showed higher Na⁺ efflux (Wang et al., 2006).Here, we wished to explore the role(s) by which ThSOS1, the SOS1 homolog in Thellungiella, could be involved in shaping the halophytic character of the species using ectopic expression of the gene in yeast and in Arabidopsis and Thellungiella SOS1-RNA interference (RNAi) lines. The results identified ThSOS1 as a genetic element whose activity limits Na⁺ accumulation and affects the distribution of Na⁺ ions at high concentration, thus acting as a major tolerance determinant.     

Germination responses of <Emphasis Type="Italic">Erica andevalensis</Emphasis> to different chemical and physical treatments

Erica andevalensis Cabezudo & Rivera is a threatened edaphic endemic species of Andalusia (SW Spain). Under natural conditions, the plants produce a very large number of small seeds (0.3–0.4 mm) but very few seedlings survive. Different treatments (high temperature, cold pre-treatment, nitrogen salts, and gibberellic acid applications) were tested to assess germination patterns in different populations and to determinate the most favorable conditions for germination. Gibberellic acid was provided in five different concentrations from 0 to 400 ppm GA₃, while nitrogen was applied as 10 mM of either KNO₃ or NH₄NO₃. The effect of pH on germination was also tested. The species always showed a low germination rate (6.50–22%) that was not stimulated either by 1 or 4 months in dry cold pre-treatment, nitrogen application, acid pH medium, or by high temperature (80°C for 10 min); although gibberellic acid application (100–400 ppm) significantly enhanced germination. The highest percentage of germination (41.6%) was achieved with a mean germination time to start germination (t ₀) of 7.6 ± 0.54 days when the seeds were subjected to 400 ppm gibberellic acid treatment. The population origin did not have a significant effect on germination percentage.     

The AtNHX1 exchanger mediates potassium compartmentation in vacuoles of transgenic tomato

NHX‐type antiporters in the tonoplast have been reported to increase the salt tolerance of various plants species, and are thought to mediate the compartmentation of Na⁺ in vacuoles. However, all isoforms characterized so far catalyze both Na⁺/H⁺ and K⁺/H⁺ exchange. Here, we show that AtNHX1 has a critical involvement in the subcellular partitioning of K⁺, which in turn affects plant K⁺ nutrition and Na⁺ tolerance. Transgenic tomato plants overexpressing AtNHX1 had larger K⁺ vacuolar pools in all growth conditions tested, but no consistent enhancement of Na⁺ accumulation was observed under salt stress. Plants overexpressing AtNHX1 have a greater capacity to retain intracellular K⁺ and to withstand salt‐shock. Under K⁺‐limiting conditions, greater K⁺ compartmentation in the vacuole occurred at the expense of the cytosolic K⁺ pool, which was lower in transgenic plants. This caused the early activation of the high‐affinity K⁺ uptake system, enhanced K⁺ uptake by roots, and increased the K⁺ content in plant tissues and the xylem sap of transformed plants. Our results strongly suggest that NHX proteins are likely candidates for the H⁺‐linked K⁺ transport that is thought to facilitate active K⁺ uptake at the tonoplast, and the partitioning of K⁺ between vacuole and cytosol.