Natural variants of AtHKT1 enhance Na+ accumulation in two wild populations of Arabidopsis

Plants are sessile and therefore have developed mechanisms to adapt to their environment, including the soil mineral nutrient composition. Ionomics is a developing functional genomic strategy designed to rapidly identify the genes and gene networks involved in regulating how plants acquire and accumulate these mineral nutrients from the soil. Here, we report on the coupling of high-throughput elemental profiling of shoot tissue from various Arabidopsis accessions with DNA microarray-based bulk segregant analysis and reverse genetics, for the rapid identification of genes from wild populations of Arabidopsis that are involved in regulating how plants acquire and accumulate Na(+) from the soil. Elemental profiling of shoot tissue from 12 different Arabidopsis accessions revealed that two coastal populations of Arabidopsis collected from Tossa del Mar, Spain, and Tsu, Japan (Ts-1 and Tsu-1, respectively), accumulate higher shoot levels of Na(+) than do Col-0 and other accessions. We identify AtHKT1, known to encode a Na(+) transporter, as being the causal locus driving elevated shoot Na(+) in both Ts-1 and Tsu-1. Furthermore, we establish that a deletion in a tandem repeat sequence approximately 5 kb upstream of AtHKT1 is responsible for the reduced root expression of AtHKT1 observed in these accessions. Reciprocal grafting experiments establish that this loss of AtHKT1 expression in roots is responsible for elevated shoot Na(+). Interestingly, and in contrast to the hkt1-1 null mutant, under NaCl stress conditions, this novel AtHKT1 allele not only does not confer NaCl sensitivity but also cosegregates with elevated NaCl tolerance. We also present all our elemental profiling data in a new open access ionomics database, the Purdue Ionomics Information Management System (PiiMS; http://www.purdue.edu/dp/ionomics). Using DNA microarray-based genotyping has allowed us to rapidly identify AtHKT1 as the casual locus driving the natural variation in shoot Na(+) accumulation we observed in Ts-1 and Tsu-1. Such an approach overcomes the limitations imposed by a lack of established genetic markers in most Arabidopsis accessions and opens up a vast and tractable source of natural variation for the identification of gene function not only in ionomics but also in many other biological processes.     

AtHKT1 facilitates Na+ homeostasis and K+ nutrition in planta

Genetic and physiological data establish that Arabidopsis AtHKT1 facilitates Na(+) homeostasis in planta and by this function modulates K(+) nutrient status. Mutations that disrupt AtHKT1 function suppress NaCl sensitivity of sos1-1 and sos2-2, as well as of sos3-1 seedlings grown in vitro and plants grown in controlled environmental conditions. hkt1 suppression of sos3-1 NaCl sensitivity is linked to higher Na(+) content in the shoot and lower content of the ion in the root, reducing the Na(+) imbalance between these organs that is caused by sos3-1. AtHKT1 transgene expression, driven by its innate promoter, increases NaCl but not LiCl or KCl sensitivity of wild-type (Col-0 gl1) or of sos3-1 seedlings. NaCl sensitivity induced by AtHKT1 transgene expression is linked to a lower K(+) to Na(+) ratio in the root. However, hkt1 mutations increase NaCl sensitivity of both seedlings in vitro and plants grown in controlled environmental conditions, which is correlated with a lower K(+) to Na(+) ratio in the shoot. These results establish that AtHKT1 is a focal determinant of Na(+) homeostasis in planta, as either positive or negative modulation of its function disturbs ion status that is manifested as salt sensitivity. K(+)-deficient growth of sos1-1, sos2-2, and sos3-1 seedlings is suppressed completely by hkt1-1. AtHKT1 transgene expression exacerbates K(+) deficiency of sos3-1 or wild-type seedlings. Together, these results indicate that AtHKT1 controls Na(+) homeostasis in planta and through this function regulates K(+) nutrient status.     

The Arabidopsis HKT1 gene homolog mediates inward Na(+) currents in xenopus laevis oocytes and Na(+) uptake in Saccharomyces cerevisiae

The Na(+)-K(+) co-transporter HKT1, first isolated from wheat, mediates high-affinity K(+) uptake. The function of HKT1 in plants, however, remains to be elucidated, and the isolation of HKT1 homologs from Arabidopsis would further studies of the roles of HKT1 genes in plants. We report here the isolation of a cDNA homologous to HKT1 from Arabidopsis (AtHKT1) and the characterization of its mode of ion transport in heterologous systems. The deduced amino acid sequence of AtHKT1 is 41% identical to that of HKT1, and the hydropathy profiles are very similar. AtHKT1 is expressed in roots and, to a lesser extent, in other tissues. Interestingly, we found that the ion transport properties of AtHKT1 are significantly different from the wheat counterpart. As detected by electrophysiological measurements, AtHKT1 functioned as a selective Na(+) uptake transporter in Xenopus laevis oocytes, and the presence of external K(+) did not affect the AtHKT1-mediated ion conductance (unlike that of HKT1). When expressed in Saccharomyces cerevisiae, AtHKT1 inhibited growth of the yeast in a medium containing high levels of Na(+), which correlates to the large inward Na(+) currents found in the oocytes. Furthermore, in contrast to HKT1, AtHKT1 did not complement the growth of yeast cells deficient in K(+) uptake when cultured in K(+)-limiting medium. However, expression of AtHKT1 did rescue Escherichia coli mutants carrying deletions in K(+) transporters. The rescue was associated with a less than 2-fold stimulation of K(+) uptake into K(+)-depleted cells. These data demonstrate that AtHKT1 differs in its transport properties from the wheat HKT1, and that AtHKT1 can mediate Na(+) and, to a small degree, K(+) transport in heterologous expression systems.     

Enhanced salt tolerance mediated by AtHKT1 transporter-induced Na unloading from xylem vessels to xylem parenchyma cells

AtHKT1 is a sodium (Na+) transporter that functions in mediating tolerance to salt stress. To investigate the membrane targeting of AtHKT1 and its expression at the translational level, antibodies were generated against peptides corresponding to the first pore of AtHKT1. Immunoelectron microscopy studies using anti-AtHKT1 antibodies demonstrate that AtHKT1 is targeted to the plasma membrane in xylem parenchyma cells in leaves. AtHKT1 expression in xylem parenchyma cells was also confirmed by AtHKT1 promoter-GUS reporter gene analyses. Interestingly, AtHKT1 disruption alleles caused large increases in the Na+ content of the xylem sap and conversely reduced the Na+ content of the phloem sap. The athkt1 mutant alleles had a smaller and inverse influence on the potassium (K+) content compared with the Na+ content of the xylem, suggesting that K+ transport may be indirectly affected. The expression of AtHKT1 was modulated not only by the concentrations of Na+ and K+ but also by the osmolality of non-ionic compounds. These findings show that AtHKT1 selectively unloads sodium directly from xylem vessels to xylem parenchyma cells. AtHKT1 mediates osmolality balance between xylem vessels and xylem parenchyma cells under saline conditions. Thus AtHKT1 reduces the sodium content in xylem vessels and leaves, thereby playing a central role in protecting plant leaves from salinity stress.     

Functional analysis of AtHKT1 in Arabidopsis shows that Na(+) recirculation by the phloem is crucial for salt tolerance

Two allelic recessive mutations of Arabidopsis, sas2-1 and sas2-2, were identified as inducing sodium overaccumulation in shoots. The sas2 locus was found (by positional cloning) to correspond to the AtHKT1 gene. Expression in Xenopus oocytes revealed that the sas2-1 mutation did not affect the ionic selectivity of the transporter but strongly reduced the macro scopic (whole oocyte current) transport activity. In Arabidopsis, expression of AtHKT1 was shown to be restricted to the phloem tissues in all organs. The sas2-1 mutation strongly decreased Na(+) concentration in the phloem sap. It led to Na(+) overaccumulation in every aerial organ (except the stem), but to Na(+) underaccumulation in roots. The sas2 plants displayed increased sensitivity to NaCl, with reduced growth and even death under moderate salinity. The whole set of data indicates that AtHKT1 is involved in Na(+) recirculation from shoots to roots, probably by mediating Na(+) loading into the phloem sap in shoots and unloading in roots, this recirculation removing large amounts of Na(+) from the shoot and playing a crucial role in plant tolerance to salt.     

Soil bacteria confer plant salt tolerance by tissue-specific regulation of the sodium transporter HKT1

Elevated sodium (Na(+)) decreases plant growth and, thereby, agricultural productivity. The ion transporter high-affinity K(+) transporter (HKT)1 controls Na(+) import in roots, yet dysfunction or overexpression of HKT1 fails to increase salt tolerance, raising questions as to HKT1's role in regulating Na(+) homeostasis. Here, we report that tissue-specific regulation of HKT1 by the soil bacterium Bacillus subtilis GB03 confers salt tolerance in Arabidopsis thaliana. Under salt stress (100 mM NaCl), GB03 concurrently down- and upregulates HKT1 expression in roots and shoots, respectively, resulting in lower Na(+) accumulation throughout the plant compared with controls. Consistent with HKT1 participation in GB03-induced salt tolerance, GB03 fails to rescue salt-stressed athkt1 mutants from stunted foliar growth and elevated total Na(+) whereas salt-stressed Na(+) export mutants sos3 show GB03-induced salt tolerance with enhanced shoot and root growth as well as reduced total Na(+). These results demonstrate that tissue-specific regulation of HKT1 is critical for managing Na(+) homeostasis in salt-stressed plants, as well as underscore the breadth and sophistication of plant-microbe interactions.     

A genomic selection component analysis characterizes migration‐selection balance

The genetic differentiation of populations in response to local selection pressures has long been studied by evolutionary biologists, but key details about the process remain obscure. How rapidly can local adaptation evolve, how extensive is the process across the genome, and how strong are the opposing forces of natural selection and gene flow? Here, we combine direct measurement of survival and reproduction with whole‐genome genotyping of a plant species (Mimulus guttatus) that has recently invaded a novel habitat (the Quarry population). We renovate the classic selection component method to accommodate genomic data and observe selection at SNPs throughout the genome. SNPs showing viability selection in Quarry exhibit elevated divergence from neighboring populations relative to neutral SNPs. We also find that nonsignificant SNPs exhibit a subtle, but still significant, change in allele frequency toward neighboring populations, a predicted effect of gene flow. Given that the Quarry population is most probably only 30–40 generations old, the alleles conferring local advantage are almost certainly older than the population itself. Thus, local adaptation owes to the recruitment of standing genetic variation.     

Altered shoot/root Na+ distribution and bifurcating salt sensitivity in Arabidopsis by genetic disruption of the Na+ transporter AtHKT1

Sodium (Na+) is toxic to most plants, but the molecular mechanisms of plant Na+ uptake and distribution remain largely unknown. Here we analyze Arabidopsis lines disrupted in the Na+ transporter AtHKT1. AtHKT1 is expressed in the root stele and leaf vasculature. athkt1 null plants exhibit lower root Na+ levels and are more salt resistant than wild-type in short-term root growth assays. In shoot tissues, however, athkt1 disruption produces higher Na+ levels, and athkt1 and athkt1/sos3 shoots are Na+-hypersensitive in long-term growth assays. Thus wild-type AtHKT1 controls root/shoot Na+ distribution and counteracts salt stress in leaves by reducing leaf Na+ accumulation.     

Genetics of drought adaptation in Arabidopsis thaliana II. QTL analysis of a new mapping population, KAS-1 x TSU-1

Despite compelling evidence that adaptation to local climate is common in plant populations, little is known about the evolutionary genetics of traits that contribute to climatic adaptation. A screen of natural accessions of Arabidopsis thaliana revealed Tsu-1 and Kas-1 to be opposite extremes for water-use efficiency and climate at collection sites for these accessions differs greatly. To provide a tool to understand the genetic basis of this putative adaptation, Kas-1 and Tsu-1 were reciprocally crossed to create a new mapping population. Analysis of F(3) families showed segregating variation in both delta(13)C and transpiration rate, and as expected these traits had a negative genetic correlation (r(g)=- 0.3). 346 RILs, 148 with Kas-1 cytoplasm and 198 with Tsu-1 cytoplasm, were advanced to the F(9) and genotyped using 48 microsatellites and 55 SNPs for a total of 103 markers. This mapping population was used for QTL analysis of delta(13)C using F(9) RIL means. Analysis of this reciprocal cross showed a large effect of cytoplasmic background, as well as two QTL for delta(13)C. The Kas-1 x Tsu-1 mapping population provides a powerful new resource for mapping QTL underlying natural variation and for dissecting the genetic basis of water-use efficiency differences.     

Variation in resistance and virulence in the interaction between Arabidopsis thaliana and a bacterial pathogen

We examined patterns of variation and the extent of local adaptation in the interaction between the highly selfing annual weed Arabidopsis thaliana and its foliar bacterial pathogen Pseudomonas viridiflava by cross-infecting 23 bacterial isolates with 35 plant lines collected from six fallow or cultivated fields in the Midwest, USA. We used two measures of resistance and virulence: bacterial count in the leaf and symptom development four days after infection. We found variation in resistance in A. thaliana and virulence in P. viridiflava, as well as a significant difference in symptoms between two distinct genetic clades within P. viridiflava. We also observed that both resistance and plant development rate varied with field type of origin (cultivated or fallow), possibly through age-related resistance, a developmentally regulated general form of resistance. Finally, we did not observe local adaptation by host or pathogen, rather we found patterns of variation across populations that depended in part on P. viridiflava clade. These data suggest that the interaction between A. thaliana and P. viridiflava varies across space and is mediated by the selection regime of the host populations and differential performance of the P. viridiflava clades. This is one of a very limited number of studies examining a bacterial pathogen of wild plant populations and one of a few studies to examine patterns of variation in a plant-pathogen association that is not a highly specialized gene-for-gene interaction.     

Temporal variation in genetic diversity and effective population size of Mediterranean and subalpine Arabidopsis thaliana populations

Currently, there exists a limited knowledge on the extent of temporal variation in population genetic parameters of natural populations. Here, we study the extent of temporal variation in population genetics by genotyping 151 genome-wide SNP markers polymorphic in 466 individuals collected from nine populations of the annual plant Arabidopsis thaliana during 4 years. Populations are located along an altitudinal climatic gradient from Mediterranean to subalpine environments in NE Spain, which has been shown to influence key demographic attributes and life cycle adaptations. Genetically, A. thaliana populations were more variable across space than over time. Common multilocus genotypes were detected several years in the same population, whereas low-frequency multilocus genotypes appeared only 1 year. High-elevation populations were genetically poorer and more variable over time than low-elevation populations, which might be caused by a higher overall demographic instability at higher altitudes. Estimated effective population sizes were low but also showed a significant decreasing trend with increasing altitude, suggesting a deeper impact of genetic drift at high-elevation populations. In comparison with single-year samplings, repeated genotyping over time captured substantially higher amount of genetic variation contained in A. thaliana populations. Furthermore, repeated genotyping of populations provided novel information on the genetic properties of A. thaliana populations and allowed hypothesizing on their underlying mechanisms. Therefore, including temporal genotyping programmes into traditional population genetic studies can significantly increase our understanding of the dynamics of natural populations.     

Regulated AtHKT1 gene expression by a distal enhancer element and DNA methylation in the promoter plays an important role in salt tolerance

Through sos3 (salt overly sensitive 3) suppressor screening, two allelic suppressor mutants that are weak alleles of the strong sos3 suppressor sos3hkt1-1 were recovered. Molecular characterization identified T-DNA insertions in the distal promoter region of the Arabidopsis thaliana HKT1 (AtHKT1, At4g10310) in these two weak sos3 suppressors, which results in physical separation of a tandem repeat from the proximal region of the AtHKT1 promoter. The tandem repeat is approximately 3.9 kb upstream of the ATG start codon and functions as an enhancer element to promote reporter gene expression. A putative small RNA target region about 2.6 kb upstream of the ATG start codon is heavily methylated. CHG and CHH methylation but not CG methylation is significantly reduced in the small RNA biogenesis mutant rdr2, indicating that non-CG methylation in this region is mediated by small RNAs. Analysis of AtHKT1 expression in rdr2 suggests that non-CG methylation in the putative small RNA target region represses AtHKT1 expression in shoots. The DNA methylation-deficient mutant met1-3 has nearly complete loss of total cytosine methylation in the putative small RNA target region and is hypersensitive to salt stress. The putative small RNA target region and the tandem repeat are essential for maintaining AtHKT1 expression patterns crucial for salt tolerance.     

The Na+ transporter AtHKT1;1 controls retrieval of Na+ from the xylem in Arabidopsis

HKT-type transporters appear to play key roles in Na(+) accumulation and salt sensitivity in plants. In Arabidopsis HKT1;1 has been proposed to influx Na(+) into roots, recirculate Na(+) in the phloem and control root : shoot allocation of Na(+). We tested these hypotheses using (22)Na(+) flux measurements and ion accumulation assays in an hkt1;1 mutant and demonstrated that AtHKT1;1 contributes to the control of both root accumulation of Na(+) and retrieval of Na(+) from the xylem, but is not involved in root influx or recirculation in the phloem. Mathematical modelling indicated that the effects of the hkt1;1 mutation on root accumulation and xylem retrieval were independent. Although AtHKT1;1 has been implicated in regulation of K(+) transport and the hkt1;1 mutant showed altered net K(+) accumulation, (86)Rb(+) uptake was unaffected by the hkt1;1 mutation. The hkt1;1 mutation has been shown previously to rescue growth of the sos1 mutant on low K(+); however, HKT1;1 knockout did not alter K(+) or (86)Rb(+) accumulation in sos1.     

Inter-annual and spatial climatic variability have led to a balance between local fluctuating selection and wide-range directional selection in a perennial grass species

Background and AimsThe persistence of a plant population under a specific local climatic regime requires phenotypic adaptation with underlying particular combinations of alleles at adaptive loci. The level of allele diversity at adaptive loci within a natural plant population conditions its potential to evolve, notably towards adaptation to a change in climate. Investigating the environmental factors that contribute to the maintenance of adaptive diversity in populations is thus worthwhile. Within-population allele diversity at adaptive loci can be partly driven by the mean climate at the population site but also by its temporal variability.MethodsThe effects of climate temporal mean and variability on within-population allele diversity at putatively adaptive quantitative trait loci (QTLs) were evaluated using 385 natural populations of Lolium perenne (perennial ryegrass) collected right across Europe. For seven adaptive traits related to reproductive phenology and vegetative potential growth seasonality, the average within-population allele diversity at major QTLs (HeA) was computed.Key ResultsSignificant relationships were found between HeA of these traits and the temporal mean and variability of the local climate. These relationships were consistent with functional ecology theory.ConclusionsResults indicated that temporal variability of local climate has likely led to fluctuating directional selection, which has contributed to the maintenance of allele diversity at adaptive loci and thus potential for further adaptation.     

NKS1, Na(+)- and K(+)-sensitive 1, regulates ion homeostasis in an SOS-independent pathway in Arabidopsis

An Arabidopsis thaliana mutant, nks1-1, exhibiting enhanced sensitivity to NaCl was identified in a screen of a T-DNA insertion population in the genetic background of Col-0 gl1sos3-1. Analysis of the genome sequence in the region flanking the T-DNA left border indicated two closely linked mutations in the gene encoded at locus At4g30996. A second allele, nks1-2, was obtained from the Arabidopsis Biological Resource Center. NKS1 mRNA was detected in all parts of wild-type plants but was not detected in plants of either mutant, indicating inactivation by the mutations. Both mutations in NKS1 were associated with increased sensitivity to NaCl and KCl, but not to LiCl or mannitol. NaCl sensitivity was associated with nks1 mutations in Arabidopsis lines expressing either wild type or null alleles of SOS1, SOS2 or SOS3. The NaCl-sensitive phenotype of the nks1-2 mutant was complemented by expression of a full-length NKS1 allele from the CaMV35S promoter. When grown in medium containing NaCl, nks1 mutants accumulated more Na(+) than wild type and K(+)/Na(+) homeostasis was perturbed. It is proposed NKS1, a plant-specific gene encoding a 19kDa endomembrane-localized protein of unknown function, is part of an ion homeostasis regulation pathway that is independent of the SOS pathway.     

Salinity Adaptation and the Contribution of Parental Environmental Effects in Medicago truncatula

High soil salinity negatively influences plant growth and yield. Some taxa have evolved mechanisms for avoiding or tolerating elevated soil salinity, which can be modulated by the environment experienced by parents or offspring. We tested the contribution of the parental and offspring environments on salinity adaptation and their potential underlying mechanisms. In a two-generation greenhouse experiment, we factorially manipulated salinity concentrations for genotypes of Medicago truncatula that were originally collected from natural populations that differed in soil salinity. To compare population level adaptation to soil salinity and to test the potential mechanisms involved we measured two aspects of plant performance, reproduction and vegetative biomass, and phenological and physiological traits associated with salinity avoidance and tolerance. Saline-origin populations had greater biomass and reproduction under saline conditions than non-saline populations, consistent with local adaptation to saline soils. Additionally, parental environmental exposure to salt increased this difference in performance. In terms of environmental effects on mechanisms of salinity adaptation, parental exposure to salt spurred phenological differences that facilitated salt avoidance, while offspring exposure to salt resulted in traits associated with greater salt tolerance. Non-saline origin populations expressed traits associated with greater growth in the absence of salt while, for saline adapted populations, the ability to maintain greater performance in saline environments was also associated with lower growth potential in the absence of salt. Plastic responses induced by parental and offspring environments in phenology, leaf traits, and gas exchange contribute to salinity adaptation in M. truncatula. The ability of plants to tolerate environmental stress, such as high soil salinity, is likely modulated by a combination of parental effects and within-generation phenotypic plasticity, which are likely to vary in populations from contrasting environments.     

AtHKT1;1 mediates nernstian sodium channel transport properties in Arabidopsis root stelar cells

The Arabidopsis AtHKT1;1 protein was identified as a sodium (Na⁺) transporter by heterologous expression in Xenopus laevis oocytes and Saccharomyces cerevisiae. However, direct comparative in vivo electrophysiological analyses of a plant HKT transporter in wild-type and hkt loss-of-function mutants has not yet been reported and it has been recently argued that heterologous expression systems may alter properties of plant transporters, including HKT transporters. In this report, we analyze several key functions of AtHKT1;1-mediated ion currents in their native root stelar cells, including Na⁺ and K⁺ conductances, AtHKT1;1-mediated outward currents, and shifts in reversal potentials in the presence of defined intracellular and extracellular salt concentrations. Enhancer trap Arabidopsis plants with GFP-labeled root stelar cells were used to investigate AtHKT1;1-dependent ion transport properties using patch clamp electrophysiology in wild-type and athkt1;1 mutant plants. AtHKT1;1-dependent currents were carried by sodium ions and these currents were not observed in athkt1;1 mutant stelar cells. However, K⁺ currents in wild-type and athkt1;1 root stelar cell protoplasts were indistinguishable correlating with the Na⁺ over K⁺ selectivity of AtHKT1;1-mediated transport. Moreover, AtHKT1;1-mediated currents did not show a strong voltage dependence in vivo. Unexpectedly, removal of extracellular Na⁺ caused a reduction in AtHKT1;1-mediated outward currents in Columbia root stelar cells and Xenopus oocytes, indicating a role for external Na⁺ in regulation of AtHKT1;1 activity. Shifting the NaCl gradient in root stelar cells showed a Nernstian shift in the reversal potential providing biophysical evidence for the model that AtHKT1;1 mediates passive Na⁺ channel transport properties.     

Control of sodium transport in durum wheat

In many species, salt sensitivity is associated with the accumulation of sodium (Na(+)) in photosynthetic tissues. Na(+) uptake to leaves involves a series of transport steps and so far very few candidate genes have been implicated in the control of these processes. In this study, Na(+) transport was compared in two varieties of durum wheat (Triticum turgidum) L. subsp. durum known to differ in salt tolerance and Na(+) accumulation; the relatively salt tolerant landrace line 149 and the salt sensitive cultivar Tamaroi. Genetic studies indicated that these genotypes differed at two major loci controlling leaf blade Na(+) accumulation (R. Munns, G.J. Rebetzke, S. Husain, R.A. James, R.A. Hare [2003] Aust J Agric Res 54: 627-635). The physiological traits determined by these genetic differences were investigated using measurements of unidirectional (22)Na(+) transport and net Na(+) accumulation. The major differences in Na(+) transport between the genotypes were (1) the rate of transfer from the root to the shoot (xylem loading), which was much lower in the salt tolerant genotype, and (2) the capacity of the leaf sheath to extract and sequester Na(+) as it entered the leaf. The genotypes did not differ significantly in unidirectional root uptake of Na(+) and there was no evidence for recirculation of Na(+) from shoots to roots. It is likely that xylem loading and leaf sheath sequestration are separate genetic traits that interact to control leaf blade Na(+).