AtHKT1 drives adaptation of Arabidopsis thaliana to salinity by reducing floral sodium content

Arabidopsis thaliana high-affinity potassium transporter 1 (AtHKT1) limits the root-to-shoot sodium transportation and is believed to be essential for salt tolerance in A. thaliana. Nevertheless, natural accessions with ‘weak allele’ of AtHKT1, e.g. Tsu-1, are mainly distributed in saline areas and are more tolerant to salinity. These findings challenge the role of AtHKT1 in salt tolerance and call into question the involvement of AtHKT1 in salinity adaptation in A. thaliana. Here, we report that AtHKT1 indeed drives natural variation in the salt tolerance of A. thaliana and the coastal AtHKT1, so-called weak allele, is actually hyper-functional in reducing flowers sodium content upon salt stress. Our data showed that AtHKT1 positively contributes to saline adaptation in a linear manner. Forward and reverse genetics analysis established that the single AtHKT1 locus is responsible for the variation in the salinity adaptation between Col-0 and Tsu-1. Reciprocal grafting experiments revealed that shoot AtHKT1 determines the salt tolerance of Tsu-1, whereas root AtHKT1 primarily drives the salt tolerance of Col-0. Furthermore, evidence indicated that Tsu-1 AtHKT1 is highly expressed in stems and is more effective compared to Col-0 AtHKT1 at limiting sodium flow to the flowers. Such efficient retrieval of sodium to the reproductive organ endows Tsu-1 with stronger fertility compared to Col-0 upon salt stress, thus improving Tsu-1 adaptation to a coastal environment. To conclude, our data not only confirm the role of AtHKT1 in saline adaptation, but also sheds light on our understanding of the salt tolerance mechanisms in plants.


Introduction
Although plants are sessile and are passively exposed to varying environments, they can successfully complete their life cycle and disperse across a range of environmental conditions. These capabilities are attributable to their physiological plastic and complex gene regulation networks, as well as to their affluent genetic diversities. Therefore, revealing the genetic variation that drives adaptation to particular environments is critical for understanding natural selection and evolutionary mechanisms. Numerous studies have elucidated the genetic changes in different plant species in their adaptation to extreme temperature [1], flowering time [2], photoperiod [3], and microbial attack [4]. Soil is a key environmental factor in maintaining plant growth and a major driving force in plant evolution. Nevertheless, little progress has been made in determining the genetic basis underlying plant adaptation to different edaphic conditions. Sodium is a non-essential element that is toxic to most plants. Saline soil is an important driver of genetic variation. A previous study has shown that genome duplication alters fitness to allow plants to adapt to saline environments [5]. Nonetheless, only very limited evidence supports the involvement of a specific gene in local adaptation to saline habitats.
HKT1 (High-affinity Potassium Transporter) was first isolated from wheat and was shown to function as a K + /Na + symporter [6]. Its homologs in various species, such as AtHKT1, OsHKT1;1 and SKC1/OsHKT1;5, are generally expressed in xylems of different organs and contribute to plant salt tolerance by unloading sodium from the transpiration stream [7][8][9][10][11]. Interestingly, natural variation in these HKT homologs has been observed in both A. thaliana and rice populations. Four amino acid substitutions in SKC1/OsHKT1;5 significantly increase its sodium transport activity, restricting sodium flux to leaves and greatly enhancing the salt tolerance of the salt-tolerant rice variety Nona Bokra [9].
A. thaliana is widely distributed in all kinds of geographical environments, and many ecotypes have adapted to saline conditions [12]. Tsu-1 and Ts-1 are two coastal A. thaliana accessions that are highly tolerant to salt stress [13]. However, unlike the salt-tolerant rice variety Nona Bokra, they accumulate very high sodium in their leaves. Genetic analysis has revealed that the high leaf sodium phenotypes of Tsu-1 and Ts-1 are controlled by the AtHKT1 locus [13]. AtHKT1 is expressed at extremely low levels in the roots of these two accessions due to the absence of a distal enhancer [14]. Genome-wide association studies (GWAS) have confirmed that AtHKT1 controls the natural variation of leaf sodium content in A. thaliana populations worldwide and have revealed that A. thaliana accessions with weak AtHKT1 alleles are predominantly distributed in coastal or saline areas [15]. These results, together with other molecular ecology evidence, suggest that AtHKT1 plays a negative role in the salinity adaptation of A. thaliana [12]. However, this conclusion seemingly conflicts with the observation that the null hkt1-1 mutant is hypersensitive to salt stress [16][17][18]. Solving this puzzle is particularly important because it may uncover the role of AtHKT1 in local adaptation, improve our knowledge of the salt tolerance mechanisms of plants and help resolve the long debate about the relationship between shoot sodium content and salt tolerance.
Edaphic salinity threatens plants via osmotic stress and ion toxicity. Halophyte plants generally accumulate sodium in their shoots, as high sodium helps cells resist osmotic stress, and the sodium is not toxic to the cell as long as it is sequestered in the vacuole [19]. Although A. thaliana is not a halophyte, the low expression of AtHKT1 and the high leaf sodium phenotype of coastal salt-tolerant accessions might suggest that A. thaliana may use a strategy similar to halophytes to adapt to saline habitats. It was therefore hypothesized that AtHKT1 might serve as a double-edged sword for salt tolerance, while weak expression of AtHKT1 could well balance the osmotic and ionic toxicity of high salt for the best salt tolerance [13,15]. Alternatively, AtHKT1 might contribute to saline adaptation in an uncharacterized mechanism. Otherwise, it should be independent of the saline adaptation.
Seed production is a key index used for evaluating crops economic efficiency in agriculture and is an essential indicator in assessing the evolutionary adaptation of plants [20]. Therefore, in this study, we used seed yield as a central index for estimating the adaptability of plants. We found that AtHKT1 positively drives plant adaptation to a coastal environment, by changing its expression pattern to efficiently retrieve sodium from the stem-to-flower xylem sap and thus reduce floral sodium content to increase fecundity upon salt stress.

Salt tolerance of A. thaliana has a linear positive relationship with expression levels of AtHKT1
To examine if there is an expression balance of AtHKT1 for the best salt tolerance, independent knockdown lines of AtHKT1 in the Col-0 background were established. We also isolated an athkt1 null mutant in the Col-0 background as a positive control by backcrossing hkt1-1 with Col-0. Under normal condition, no difference on fecundity was observed among different genotypes (Fig 1A and 1B). Though the coastal saline-adapted accession Tsu-1 produced twice as many seeds as Col-0, whereas the athkt1 knockout mutant yielded almost no seeds when they were treated with 100 mM NaCl, a mild salt stress condition that mimics the natural saline habitat of coastal A. thaliana accessions (Fig 1A and 1C). These data indicate that AtHKT1 is essential for salt tolerance and that Tsu-1 is more adapted to saline soil. Moreover, the number of seeds in AtHKT1 knockdown lines declined with decreases in AtHKT1 levels, while those expressing AtHKT1 to a similar level as Tsu-1 produced a similar number of seeds as athkt1 (Fig 1A, 1C and 1D). In addition, we found that the seed number is positively and linearly correlated with AtHKT1 expression when exposed to salt stress (Fig 1F), but not under normal condition (Fig 1E). To sum up, these data demonstrate that AtHKT1 linearly contributes to salt tolerance, and that weak expression of AtHKT1 does not really balance the osmotic and ionic toxicities for the best salt tolerance.
AtHKT1 is responsible for the saline adaptation phenotype of Tsu-1 A rough mapping has shown that the salt tolerance phenotype of Tsu-1 is linked to the AtHKT1 locus [13]. Nonetheless, it remained unclear whether AtHKT1 itself, or a locus linked to AtHKT1, controls the phenotype. To address this question, we tried to map the responsible locus or loci by QTL analysis. Epistatic interaction and additive effects generally cause a big problem for mapping QTLs. Fixing genotype of one causal locus could remove or minimize these problems, therefore we synthesized an F2 population derived from a cross between Tsu-1 and athkt1, as both alleles are hypo-functional.
We first examined the productivity of the F1 progeny and observed that F1 plants produced an amount of seeds similar to Tsu-1 when exposed to 100 mM NaCl (Fig 2A), suggesting that the adaptive trait is probably controlled by one or more dominant loci. An analysis of 201 F2 individuals further confirmed that a single major locus controls saline adaptation, given that the ratio of salt tolerant F2s (153 individuals) and salt sensitive F2s (48 individuals) conforms to 3:1 (Χ 2 = 0.08<Χ 2 0.05 = 3.84) (Fig 2B). To identify the locus, we performed a QTL analysis using 36 polymorphic genetic markers covering all five chromosomes. As expected, we identified a single major QTL on chromosome 4 that contributed to 53.1% of the variance in seed yield under salt stress ( Fig 2C). Interestingly, this candidate region contains AtHKT1, but none of the NHX genes that could sequester sodium in the vacuole [21].
We employed an enlarged F2 population with 537 individuals and developed 10 polymorphic markers in the candidate region for fine mapping. Based on genotype and phenotype analyses of the F2 individuals or their F3 progenies, we narrowed down the causal gene to a 100-kb region between genetic markers GM635 and GM645 (Fig 2D). This region contains only 26 annotated genes; and only AtHKT1 is related to salt tolerance (S1 Table), suggesting that the AtHKT1 is the causal locus for adaptation of Tsu-1 to saline conditions. To verify this, we knocked out AtHKT1 in Tsu-1 using CRISPR (clustered regulatory interspaced short palindromic repeats)-Cas9 (CRISPR-associated protein 9) technology [22]. In the T2 generation, we obtained two homologous knockout mutants of AtHKT1 and named them hkt1-c1 and hkt1-c2 (c stands for alleles generated by CRISPR-Cas9 technology). In hkt1-c1, AtHKT1 has a thymine inserted between the 450 th and 451 st nucleotides of the coding sequence (S1A Fig), while, in the hkt1-c2, four nucleotides (from the 447 th to the 450 th nucleotides after the start codon) are deleted (S1B Fig), both of which result in frame shifts.
Under normal conditions, there were no differences on seed production among hkt1-c1, hkt1-c2 and Tsu-1 (S1C Fig). Yet, when treated with 100 mM NaCl, the two mutants were totally sterile, whereas the wild control Tsu-1 was still productive (S1D Fig). The phenotypes of the two mutants were comparable to or even more severe than that of athkt1, as athkt1 still produced some seeds, although considerably less than Col-0 under the same conditions ( Fig  3A and 3B). Considering that Tsu-1 produced significantly more seeds than Col-0 in the same salt treatment experiment (Fig 3A and 3B), we conjectured that AtHKT1 is responsible for the natural variation in saline adaptation between Tsu-1 and Col-0. To further confirm that the phenotypes of hkt1-c1 and hkt1-c2 were caused by AtHKT1 knock-out, we crossed them with athkt1 respectively. The F1 plants were all sterile when treated with 100 mM NaCl (S2A and S2B Fig), verifying that the phenotypes of hkt1-c1 and hkt1-c2 were not an off-target effect.
To further determine whether a single AtHKT1 locus is sufficient to drive the variation in the salinity adaptation of A. thaliana, we cloned the genomic DNA of AtHKT1, including its long promoter from Col-0 and Tsu-1, and introduced each construct into the null athkt1 mutant separately. We tested the salt tolerance of three independent transgenic lines for each construct by assessing productivity. Transgenic lines with either the Col-0 AtHKT1 fragment or the Tsu-1 fragment could complement the salt-hypersensitive phenotype of athkt1 ( Fig 3C). Nevertheless, the Col-0 AtHKT1 fragment could only restore the saline adaptability of athkt1 to the Col-0 level, whereas the Tsu-1 fragment was sufficient to restore athkt1 to the Tsu-1 level of adaptability ( Fig 3D). These data demonstrated that a single AtHKT1 locus is sufficient to drive the natural variation in the salinity adaptation of Col-0 and Tsu-1.
Reciprocal grafting reveals that the salt tolerance phenotype of Tsu-1 is driven by shoot AtHKT1, whereas that of Col-0 is driven by root AtHKT1 Root AtHKT1 expression drives leaf sodium content in both Col-0 and Tsu-1 [13]. Nevertheless, it was unclear in what tissue AtHKT1 drives salt tolerance in these ecotypes. To address this question, we performed two reciprocal grafting experiments; one between athkt1 and Col-0, and the other was between hkt1-c1 and Tsu-1. We observed that the grafted plants with athkt1 as the shoot, and Col-0 as the root, were as tolerant as self-grafted Col-0 plants; whereas the grafted plants with Col-0 as the shoot, and athkt1 as the root, were similar to self-grafted athkt1 plants ( Fig 4A and 4B). This result indicates that salt tolerance of Col-0 predominantly attributes to root AtHKT1. However, the grafting experiment between Tsu-1 and hkt1-c1 showed the opposite results. The grafted plants with Tsu-1 scion and hkt1-c1 stock produced a similar salt tolerant phenotype as Tsu-1 self-grafted plants, whereas hkt1-c1 self-grafted plants and grafted plants with hkt1-c1 scion and Tsu-1 stock were sterile upon salt stress (Fig 4C and 4D). These data showed that the shoot expression of AtHKT1 drives the saline adaptation of Tsu-1. Moreover, they suggest that although Tsu-1 AtHKT1 is hypo-functional in roots, it is hyper-functional in shoots.

Tsu-1 AtHKT1 is hyper-functional in stems due to variations in expression
The grafting results inspired us to analyze the expression of AtHKT1 in different aerial tissues. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) showed that differences in AtHKT1 expression between Tsu-1 and Col-0 depend on the tissue and the treatment. AtHKT1 was expressed at similar levels in the rosette leaves of 4-week-old of Tsu-1 and Col-0 when exposed to 100 mM NaCl for 24 h, but its expression was higher in the rosette leaves of Col-0 than of Tsu-1 under normal conditions (S3A Fig). In flowers, Tsu-1 expressed AtHKT1 at a similar level as Col-0 under normal conditions but it expressed less than Col-0 when treated with salt ( S3B Fig). Interestingly, Tsu-1 expressed significantly higher AtHKT1 in the stems, cauline leaves and siliques compared to Col-0 ( Fig 5A). When treated with salt, Tsu-1 expressed 6 times more AtHKT1 than did Col-0 in the stems, which is consistent with the hypothesis that Tsu-1 allele of AtHKT1 is hyper-functional in shoot.
To confirm the qRT-PCR results and further examine the spatial expression patterns of AtHKT1 in detail, we generated transgenic Col-0 plants carrying a β-glucuronidase (GUS) reporter driven by the long promoter of AtHKT1 from either Tsu-1 or Col-0. We examined 5 independent transgenic lines expressing pAtHKT1 Tsu-1 ::GUS and 7 independent lines expressing pAtHKT1 Col-0 ::GUS. The transgenic lines containing the same construct showed consistent GUS signals. At the seedling stage, all 5 lines expressing pAtHKT1 Tsu-1 ::GUS exhibited very weak GUS activity in the roots, whereas 6 of the 7 lines expressing pAtHKT1 Col-0 ::GUS strongly expressed GUS in the stele of the roots (Fig 5B). These observations were consistent with previous findings [14] and with our qRT-PCR results (Fig 5C), indicating that the constructs were Successfully grafted plants (n > 12) were transferred to pots for 2 weeks following 100 mM NaCl treatment twice for 2 weeks before images were taken. Data represented with mean ± SE; significant differences were determined by ANOVA. correctly expressed in Col-0 plants. Contrary to the root results, the GUS signal in stems and cauline leaves of 4-week-old of plants was much stronger in Tsu-1 than in Col-0 ( Fig 5D). This result further confirmed the qRT-PCR results.
We further examined the detailed expression site(s) of AtHKT1 in stems by hand sectioning of the pAtHKT1:GUS transgenic lines. The GUS signal driven by the Tsu-1 AtHKT1 promoter was mainly observed in the xylem of the stem (Fig 5E), indicating that Tsu-1 AtHKT1 predominantly functions in removing sodium from the flux to the flowers. By contrast, the GUS signal driven by Col-0 AtHKT1 promoter was very weak in the xylem and was observable in the phloem ( Fig 5E). These data demonstrated that variation in the AtHKT1 promoter affects not only the expression level but also the spatial specificity of AtHKT1 expression.
In addition to large insertion/deletion (indel) polymorphisms in the promoter region (S4A Fig), multiple single nucleotide polymorphisms (SNPs) were also observed in the coding sequence with several affecting the amino acid sequence (S4B Fig). To test whether the amino acid changes in AtHKT1 in Tsu-1 affect its transport activity, we expressed AtHKT1 protein from Tsu-1 and Col-0 in Xenopus laevis oocytes for electrophysiological analysis. As expected, we detected sodium transporter activity of AtHKT1 (Fig 5F), but there was no significant difference between the proteins from Col-0 and Tsu-1, indicating that the amino acid changes do not affect AtHKT1 activity. This observation is consistent with previous findings that these changed amino acids are not in conserved regions of AtHKT1 [23,24].
Tsu-1 AtHKT1 is more effective in limiting sodium accumulation in flowers exposed to salt stress The increased expression of AtHKT1 in the xylem of Tsu-1 stems suggested that Tsu-1 is more effective than Col-0 at limiting sodium flow to flower. To confirm this hypothesis, we measured the sodium content in the flowers. Under normal conditions, athkt1 accumulated 38% higher sodium in flowers than Col-0, and hkt1-c1 accumulated 46% higher than Tsu-1, but there were no significant differences between the wild-types and between the mutants (Fig 6A). Nevertheless, the sodium content in Tsu-1 flowers was only 62% of that in Col-0 flowers when exposed to salt treatment (Fig 6B). By contrast, upon salt stress, the loss of AtHKT1 function in Col-0 resulted in a 199% increase in floral sodium, whereas the loss of AtHKT1 function in Tsu-1 resulted in a 275% increase in floral sodium (Fig 6B). These data indicated that AtHKT1 in Tsu-1 stems is more effective in limitation of sodium flux to flowers than AtHKT1 in Col-0 roots. In addition, we analyzed the relationship between floral sodium content and seed number under salt stress and found these two variables to be negatively correlated (Fig 6B Embedded), demonstrating that the sodium content in flowers under salt stress is a determinant of saline adaptation.
An examination of stem sodium content provided additional evidence supporting the idea that Tsu-1 AtHKT1 is involved in sodium removal in stems. There was no significant difference in stem sodium content among Col-0, Tsu-1, athkt1 and hkt1-c1 under normal conditions ( Fig 6C). However, under salt stress, Tsu-1 accumulated 59% more sodium in stems than did hkt1-c1, whereas Col-0 accumulated 57% less stem sodium than athkt1 ( Fig 6D); these results show that AtHKT1 in Tsu-1 promotes sodium accumulation in stems and that AtHKT1 in Col-0 has the opposite effect. This observation further confirmed that Tsu-1 AtHKT1 is hyper-functional in restricting sodium flow from stems to flowers and that Col-0 AtHKT1 functions to limit sodium flow from roots to shoots, and explains why the Tsu-1 allele of AtHKT1 endows the coastal accessions with higher adaptation ability to salinity environment than the inland accessions.
Previous studies have illustrated that the leaf sodium content cross different A. thaliana accessions under normal conditions is not associated with its salinity tolerance [15]. However, the leaf sodium content under salt stress is unknown. Therefore, we also measured the leaf sodium content under normal conditions and after salt treatment. Consistent with previous studies, Tsu-1 and the two null mutants accumulated much more leaf sodium than Col-0 under normal conditions (Fig 6E). The result was similar when the plants were treated with salt, as the leaf sodium content in Tsu-1 and the two null mutants was much higher than that in Col-0 (Fig 6F).

Discussion
The salinization of irrigated land is increasingly detrimental to plant reproduction and agricultural productivity, as most plant species are sensitive to high concentrations of sodium. Understanding the genetic and molecular mechanisms underlying salt tolerance is necessary for resolving such problems. Natural variation is an important genetic resource for studying gene function, as well as evolutionary biology. Previous studies have shown that HKT family transporters are essential for natural variation in salt tolerance of rice [9,25,26]. AtHKT1 has also been found to control natural variation in leaf sodium content of A. thaliana, but it is still unclear if and how this locus contributes to saline adaptation [13,15]. In this study, we performed a series of experiments to address this question.

AtHKT1 positively contributes to saline adaptation of A. thaliana
As seed number is an essential trait for population expansion [20], we used this trait for assessing saline adaptation of plants. Previous studies have suggested that AtHKT1 might be finetuned for balancing the ionomic and osmotic stresses caused by high salt, as weak alleles of AtHKT1 might improve salt tolerance, whereas null alleles of AtHKT1 suppress salt tolerance [13]. Our observation of the linear relationship between salt tolerance and AtHKT1 expression levels argued against this hypothesis. However, our QTL analysis suggested that AtHKT1, but not additional locus, control the salt adaptability of Tsu-1. Knockout of AtHKT1 in Tsu-1 by CRISPR-Cas9 technology and complementation athkt1-1 with Tsu-1 and Col-0 AtHKT1 finally established that AtHKT1 is not only a requirement for salt tolerance of Tsu-1 but is also a predominant, if not sole locus that drives the natural variation in salinity adaptation between Col-0 and Tsu-1. Though AtHKT1 has been found to drive natural variation in leaf sodium content, this is the first direct evidence to show that AtHKT1 plays a positive role in natural selection of saline adaptive A. thaliana accessions. These genetic evidences revealed that the Tsu-1 allele of AtHKT1 is hyper-functional but not hypo-functional.
The saline adaptive accession Tsu-1 employs a more efficient mechanism of AtHKT1 for restricting sodium accumulation in flowers Our grafting experiments revealed that the saline adaptability of Tsu-1 is driven by shoot AtHKT1 expression, which is in contrast with the premise that salt tolerance of Col-0 is predominantly driven by root AtHKT1 expression. This data indicates that AtHKT1 functions differently in different accessions. GUS reporter system and qRT-PCR confirmed that Tsu-1 AtHKT1 is hyper-functional relative to Col-0 AtHKT1 due to its high expression in stems. Interestingly, histochemical staining experiment revealed that Tsu-1 AtHKT1 is predominantly expressed in xylem parenchyma cells of stems, suggesting that it functions in restricting sodium in the stems. Measurements of stem and floral sodium concentrations of different genotypes confirmed this hypothesis. Therefore, the Tsu-1 AtHKT1 functions through a new mechanism, which is to restrict sodium in the leaves and stems. This mechanism is in contrast with that of Col-0 AtHKT1 that restricts sodium in the roots.
Flower is the most important organ of the plant. However, the germ cells in flowers are very sensitive to sodium, as such cells are weak in sodium compartmentalization and detoxification due to low vacuole capacity. By contrast, leaf cells have very large vacuoles; thus, even if they accumulate a high level of sodium, it is not highly toxic if it is sequestered in the vacuole [19]. Instead, the high accumulation of sodium in leaves would facilitate resistance to osmotic stress. This difference results in flowers being much more sensitive to salt compared to leaves. Consequently, reducing the flower sodium content but not the leaf or stem sodium content is much more important for A. thaliana upon salt stress. The Tsu-1 AtHKT1 and the Col-0 AtHKT1 represent two mechanisms for restricting sodium flow to flowers; Tsu-1 AtHKT1 unloads sodium to the leaves and stems, and Col-0 unloads sodium in the roots. According to the floral sodium concentrations in Tsu-1 and Col-0, we can conclude that the Tus-1 mechanism is more effective than Col-0 mechanism in limiting sodium flow to flowers. In addition, a previous study has shown that the Col-0 mechanism could inhibit root growth upon salt stress due to its improving sodium accumulation in the roots [17]. These findings together explain why the mechanism of Tsu-1 AtHKT1 endows A. thaliana with a higher adaptation ability to saline conditions. However, we also noticed that the Col-0 mechanism was selected by most inland accessions though it represents a low efficient mechanism in salt tolerance. So far, it is hard to conclude what is the driving force behind the selection of such a mechanism, but we suspect that it might be associated with the edaphic sodium concentrations. Inland soil contains subtoxic levels of sodium, and accumulating sodium in the roots thus might be the most economical way for A. thaliana to minimize the side-effect of sodium on flower development. By contrast, the Tsu-1 mechanism involves transporting of sodium to shoot cells and sequestrating it into the vacuoles that consume much more energy. Therefore, the selection of Col-0 mechanism or Tsu-1 mechanism might reflect the sodium levels in their habitat.
The polymorphisms in the promoter region might be responsible for the divergence in expression between Col-0 AtHKT1 and Tsu-1 AtHKT1 According to a previous study, one of the tandem repeats in the distal promoter of AtHKT1 is an enhancer that promotes the expression of AtHKT1 in roots, and the absence of this distal repeat in Tsu-1 was confirmed to be responsible for the down regulation of AtHKT1 in roots [13]. However, we noted in the results of a previous study that the expression of AtHKT1 is apparently upregulated in shoots and markedly downregulated in roots in the absence of this hypothesized distal enhancer [14]. In our study, the expression of Tsu-1 AtHKT1 was significantly higher than that of Col-0 AtHKT1 in the shoots of young seedlings. This observation suggested that the second tandem repeat might not only serve as an enhancer in roots but may also function as a repressor in shoots. In this case, the high expression of AtHKT1 in Tsu-1 stems is probably caused by the deletion of the second tandem repeat. AtHKT1 has also been reported to be regulated by DNA methylation in the promoter [14]. However, we found no difference of the small RNA target site in the promoters of AtHKT1 between Tsu-1 and Col-0 (S4 Fig). Of course, we cannot exclude the possibility that other polymorphisms among the many in the promoter region control the variation in AtHKT1 expression patterns. Further research is necessary to clarify this question.

Floral sodium contents upon mild salt stress might be a better indicator of plant adaptation to saline conditions
Shoot sodium content is often used as a parameter for assessing salt tolerance of plants, but the relationship between shoot Na + and salt tolerance raises arguments. In rice, low shoot Na + is an indicator of salt tolerance [27]. A typical case is the identification of SKC1/OsHKT1;5 that reduces leaf salt content and enhances salt tolerance [9]. However, it was reported that the growth and yield of wheat upon salt stress is not correlated with sodium concentrations in leaf blade or the whole shoot [28]. Meanwhile, the salt tolerant tomato varieties accumulate higher sodium in stems and leaves than the sensitive varieties [29,30]. These findings suggest that the relationship between shoot sodium content and salt tolerance might be genetic background dependent, and therefore not stable index for assessing plant salt tolerance. This study further supports the above conclusion. In this study, we observed that the sodium levels in flowers, but not in leaves, are positively associated with salinity adaptability. This suggests that floral sodium content might be more suitable for assessing salt tolerance of plants. Of course, this does not mean the leaf sodium does not matter for salt tolerance of plants. Actually, the increased leaf sodium causes severe leaf necrosis in athkt1 null mutants when exposed to high salt conditions [16,18,31]. But upon mild salt stress, the flowering phenotype of athkt1 null mutants is more obvious than the leaf phenotype, as its leaf still has photosynthesis ability (S5A Fig), but it is almost sterile in seed production. Therefore, leaf sodium may play different roles upon different levels of salt conditions. It is worthwhile pointing out that A. thaliana is a species of glycophytes and even salt tolerant ecotypes/accessions may not successfully colonize in high salt area. Even Tsu-1 barely produces seeds when exposed to 150 mM NaCl. Therefore, mild salt stress more accurately reflects the real habitat of A. thaliana. On the other hand, the leaf necrosis in Tsu-1 caused by high salt treatment is less severe than Col-0 (S5B Fig), though Tsu-1 accumulates as much sodium as hkt1 null mutants in the leaves. This could be caused by differential expression pattern of AtHKT1 in leaves, or controlled by some other QTLs/genes.
To our knowledge, the relationship between flower sodium content and saline adaptability has never been reported. Identifying the positive relationship between the two factors provides a new angle to assess plant salt adaptability. Of course, we are not sure if this conclusion could be extended to all plant species in current stage, and further investigation is necessary to answer this question. The seed production is a most important trait of crops in agriculture. Our finding that different expression patterns and levels of AtHKT1 result in different yield upon salt stress could inspire engineering salt tolerant plants by manipulating expression pattern of HKT1. Moreover, this finding could also be translated to crops to improve their yields in saline area.

Plant material and salt treatment
Seeds of A. thaliana were surface sterilized and sown on 1/2 Murashige and Skoog (MS, Sigma-Aldrich, St. Louis, USA) medium (pH 5.7) containing 1% (w/v) sucrose, followed by stratification at 4˚C in dark for 5 days. The seedlings were transferred into pots with artificial soil in a phytotron (16 h light/8 h dark, 22˚C day/20˚C night, 80% humidity, 80 μmolÁm -2 Ás -1 ) after being grown on the plates for 7 days. Two-week-old soil-grown plants that were irrigated with 100 mM NaCl twice for two weeks and 1 additional week later plants were used for phenotyping. Four-week-old plants irrigated with 100 mM NaCl for 24 h were used for gene expression analysis and GUS staining. Each line used for salt treatment contained ! 12 plants, and every treatment included 3 biological replicates. The athkt1 mutant was isolated from an F2 population derived from a backcross between hkt1-1 (CS6531, obtained from the Arabidopsis Biological Resource Center) and Col-0.

Hydroponic culture
Seeds of A. thaliana (Tsu-1, Col-0, AtHKT1 RNAi lines and athkt1) were stratified for 5 days at 4˚C in water. The stratified seeds were sowed in punctured holes of 1.5-mL Eppendorf tube caps stuffed with 0.1% agar, and the caps were then placed on 1.5-mL Eppendorf tube containing Hoagland solution for 14 days in a chamber at 22˚C, 70% relative humidity, and light intensity of 80 μmolÁm -2 Á s -1 on 8 h light/16 h dark. The seedlings together with caps were then transferred to a big box containing Hoagland solution for growing 2 additional weeks. The culture solution was refreshed every 5 days. The roots of one-month-old seedlings were collected for qRT analysis.

QTL mapping analysis
An F2 population derived from Tsu-1 and athkt1 was used to map the causal gene of the salt tolerance of Tsu-1. The number of seeds was calculated based on an estimation of the total grain weight and 100-grain weight. A set of 36 CAPS (Cleaved Amplified Polymorphism Sequences) and SSR (Simple Sequence Repeat) markers covering the 5 chromosomes of A. thaliana were used to genotype all F2 individuals. QTL analysis was performed using the R/qtl package (http://www.rqtl.org). To narrow down the candidate region, 537 F2 individuals and 10 newly developed CAPS markers were used to fine map the target region. All CAPS and SSR markers are listed in S2 Table. Vector construction and plant transformation For the complementation experiment, a 9.3-kb AtHKT1 fragment including a 5.3-kb promoter region from Col-0 was amplified by overlap PCR using primers HKT-pro-gene L, PHMS-HKT1-MID-R, PHMS-HKT1-MID-L and HKT-pro-gene R (S3 Table). The complementation fragment for Tsu-1 AtHKT1 was amplified using the same strategy but with primers Tsu-HKT-pro-gene L, PHMS-HKT1-MID-R, PHMS-HKT1-MID-L and HKT-pro-gene R. The fragments were cloned into a binary vector, pHMS, which was modified from the pHB vector [32] by infusion recombination cloning using a Hieff Clone One-step PCR Cloning Kit (Yisheng Co. Ltm, Shanghai, China).
To construct the promoter:GUS vector, the AtHKT1 promoters were amplified from genomic DNA using primers HKT-GUSL TONG and HKT-GUSR TONG for Col-0 and primers HKT-GUSL TONG 2 and HKT-GUSR TONG for Tsu-1. The fragments were recombined into the binary vector pCAMBIA1303 using the Hieff Clone One-step PCR Cloning Kit. GUS histochemical staining was performed as described previously [9].
To construct the Col-0 AtHKT1 RNAi vector, two 184-bp fragments from the AtHKT1 fulllength cDNA sequence were amplified using the primers AtHKT1 F (PstI restriction site) and R (XbaI restriction site) for sense and AtHKT1F (SacI restriction site) and R (NotI restriction site) for antisense. Both amplified fragments were sequenced and inserted into an intermediate vector, pBluescript II SK (+/-), containing a 120-nucleotide intron from the A. thaliana RTM1 gene at the XbaI and NotI sites. Then, the hairpin fragment was sub-cloned into the binary vector pCAMBIA2301, which contained a cauliflower mosaic virus (CaMV) 35S promoter and a NOS terminator. The kanamycin resistance gene (NPT II) was used as a selective marker gene.
The construction of a CRISPR-Cas9 vector targeting AtHKT1 was performed as described previously [22]. Two oligos, AtHKT1 sgRNA4-F and sgRNA4-R, were synthesized and annealed to form a target site of 20bp in length with a requisite proto-spacer-adjacent motif, PAM (NGG) sequence at the 3' end and G at the 5' end of the sequence. The target site, containing two BbsI digestion sites, was cloned into the intermediate vector AtU6-26SK. The chimeric RNA expression cassette between KpnI and SalI was cloned into the KpnI and EcoRI sites of the pCambia1300 vector (Cambia, Canberra, Australia) together with the SalI and EcoRI fragment of the Cas9 expression cassette from the 35S-Cas9-SK vector, which was a gift from the Zhu lab [22].
The expression constructs were transformed into Agrobacterium tumefaciens strain GV3101, and were then introduced into the backgrounds as indicated using the floral dip method (26). Transgenic lines were screened on 1/2 MS medium solidified with agar containing 50 mg/L hygromycin and 1% sucrose.

Electrophysiological analysis of AtHKT1 in X. laevis oocytes
We inserted the cDNA of AtHKT1 from the Col-0 and Tsu-1 alleles into the SmaI site of the expression vector pGEMHE. The capped cRNA was transcribed using the RiboMAX Large Scale RNA Production System-T7 kit (P1300, Promega, Wisconsin, USA). The cRNA quality was verified using agarose gel electrophoresis. The concentration was determined at 260 and 280 nm and adjusted to a final concentration of 0.8 μg μl -1 . We injected freshly isolated X. laevis oocytes with 23 nL of cRNA and used the oocytes for voltage-clamp experiments 3-4 days later. Electrophysiological experiments were conducted using a two-electrode voltage clamp amplifier as described previously [9]. We bathed the oocytes in a solution containing 6 mM MgCl 2 , 1.8 mM CaCl 2 , 10 mM MES-Tris (pH 5.5), 185 mM D-mannitol and 10 mM Na-glutamate salts. The voltage protocols used are described in the figure legends.

ICP-MS analysis
The process of A. thaliana elemental analysis via inductively coupled plasma mass spectrometry (ICP-MS) has been described previously [33]. Briefly, different tissues, including flowers, whole stems and rosette leaves, from five-week-old plants treated twice with 100 mM NaCl or from untreated plants were cut with a scalpel while holding the plants with plastic tweezers. The collected tissues were rinsed with four times with 18-MO water in a 1000-mL breaker to wash off the impurities. Then, the rinsed samples were placed into a glass tube, and the samples were shifted to the bottom of the tube, making sure that no samples were left on the tube wall. The tubes were transferred to an oven at 65˚C for 12 h. After cooling, twelve samples were weighed out on an analytical balance. All the samples, including the blank controls, were digested with 1 mL of concentrated nitric acid containing an indium (In) internal standard at 115˚Cfor 4 h, and then the digested samples were diluted to 10 mL with 18 MO water. Elemental analysis for Li, B, Na, Mg, P, S, K, Ca, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Rb, Sr, Mo, and Cd was performed using an ICP-MS (NexION 350D; PerkinElmer) coupled to an Apex desolvation system and an SC-4 DX auto sampler (Elemental Scientific Inc., Omaha, NE, US). All samples were normalized to calculate weight, as determined using a heuristic algorithm using the best-measured elements, the weights of the eight weighed samples, and the solution concentrations, as previously described.

Grafting of A. thaliana plants
Seedlings were grafted as previously described [13]. Graft unions were examined under a stereoscope before transfer to potting soil to identify any adventitious root formation at or above the graft unions. Healthy grafted plants were transferred to pots and grown in a controlled environment for 2 weeks. Then, the plants were irrigated with a 100 mM NaCl solution twice, and after 2 weeks, the plants were photographed. After harvesting, the graft unions were examined again, and grafted plants with adventitious roots or without a clear graft union were excluded from subsequent analysis.

qRT-PCR
Total RNA was extracted from plants using TRNzol A+ RNA Purification reagent (TIANGEN, DP421, Beijing, China). Two micrograms of total RNA were used to synthesize first-strand cDNA with TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (Transgen, AT311-02, Beijing, China). qRT-PCR was performed using SYBR Green PCR Master Mix (TRT-101, TOYOBO, Osaka, Japan) with the first-strand cDNA as a template on a Real-Time PCR System (Bio-Rad CFX thermocycler, California, USA). Primers for qRT-PCR were designed using Primer Express Software Version 3.0 (Applied Biosystems, USA). The primers HKT1 exon2-3-1L and HKT1 exon2-3-1R were designed to span an exon-exon junction and were used to detect the gene expression level. The primers UBCF and UBCR were designed for UBIQUITIN-CONJUGATING ENZYME21 (At5g25760), which was used as the control gene. The primer sequences are shown in S3 Table. Expression data analysis was performed as described previously [34].  Fig. Allelism test of hkt1-c1, hkt1-c2 and athkt1 Writing -original draft: Dong An, Dai-Yin Chao.