Genetic Loci Associated with Early Salt Stress Responses of Roots.

Salinity is a devastating abiotic stress accounting for major crop losses yearly. Plant roots can strikingly grow away from high-salt patches. This response is termed halotropism and occurs through auxin redistribution in roots in response to a salt gradient. Here, a natural variation screen for the early and NaCl-specific halotropic response of 333 Arabidopsis accessions revealed quantitative differences in the first 24 h. These data were successfully used to identify genetic components associated with the response through Genome-Wide Association Study (GWAS). Follow-up characterization of knockout mutants in Col-0 background confirmed the role of transcription factor WRKY25, cation-proton exchanger CHX13, and a gene of unknown function DOB1 (Double Bending 1) in halotropism. In chx13 and dob1 mutants, ion accumulation and shoot biomass under salt stress were also affected. Thus, our GWAS has identified genetic components contributing to main root halotropism that provide insight into the genetic architecture underlying plant salt responses.

Introduction

Salinity has a global impact affecting about 40 million hectares of arable land and resulting in substantial crop losses yearly, since most crops are sensitive to salt (FAO and ITPS, 2015, Vargas et al., 2018). Salinity stress comprises both osmotic and ionic stress components. The presence of salt in the soil leads to decreased water potential causing a reduction of water availability for various physiological processes in the plant (Hasegawa et al., 2000, Julkowska and Testerink, 2015). The gradual build-up of ions in green tissues of plants occurs much later after salt stress exposure, resulting in further reduction in growth. When the storage capacity for sodium ions (Na+) of the plant vacuole is exceeded, increased Na+ in the cytosol competes with potassium ions (K+) and calcium ions (Ca2+) for functional binding sites of proteins leading to protein destabilization and inactivation (Maathuis, 2014, Yang and Guo, 2018).

The responses to salinity described earlier vary between and within plant species, and these responses are presumably shaped by a combination of adaptation to local environment, accumulation of mutations, gene flow between the populations, and random effects. This diversity of stress responses present within one species can be harnessed by screening natural diversity panels and subsequent Genome-Wide Association Study (GWAS) to identify genetic components of various stress responses, including salinity (Jha et al., 2010, Katori et al., 2010, Rus et al., 2006).

GWAS employs the power of natural variation in a population of certain plant species to enumerate underlying SNPs (single-nucleotide polymorphisms) responsible for certain phenotypic traits (Ogura and Busch, 2015, Weigel, 2012). It has been applied in unraveling underlying mechanisms of physiological processes and abiotic stress responses in the model species Arabidopsis. GWAS on 16 root growth traits was used to identify a Calcium Sensor receptor (CaS), determined as a regulator of main root growth rate (Slovak et al., 2014). Natural variation in rosette size of the Arabidopsis HapMap population revealed a novel candidate Leucine-Rich Repeat Kinase family protein Induced by Salt Stress (LRR-KISS), which is associated with biomass accumulation under salt stress conditions (Julkowska et al., 2016) and which was later confirmed to be required for salt stress responses (Van der Does et al., 2017). In another recent example, GWAS was performed on root traits from the HapMap collection of Arabidopsis seedlings to identify High affinity K+ transporter 1 (HKT1) and Cytochrome p450 family 79 subfamily B2 and B3 (CYP79B2/B3) as new components required for modulating lateral root development during salt stress (Julkowska et al., 2017). HKT1 was previously identified as a major factor contributing to natural variation in Na+ accumulation in the shoot of 394 Arabidopsis accessions (Baxter et al., 2010).

Despite shoot ion-toxicity becoming apparent only at later stages, Na+-specific responses have been reported to occur at early stages (<24 h) of salt stress exposure in the root. This includes an initial Ca2+ spike, modulation of the cell wall, and a salt (NaCl) avoidance response termed halotropism (Choi et al., 2014, Feng et al., 2018, Galvan-Ampudia et al., 2013, Schmöckel et al., 2015, Sun et al., 2015). Halotropism is an auxin-dependent root growth away from higher salt concentrations and toward areas with lower salt concentrations. Although previous studies identified auxin transporters to have contributory roles in root halotropic responses (Galvan-Ampudia et al., 2013, Korver et al., 2019, van den Berg et al., 2016), other components of this process remain largely unknown. Here, we screened a set of 333 Arabidopsis accessions using the robust and efficient halotropism assay and associated the observed root phenotypes with SNP markers using GWAS. This screen identified fifteen putative genes required for early halotropic responses (24 h post salt stress). For three candidate genes, encoding for a transcription factor WRKY25, a K+ transporter CHX13, and an unknown protein DOB1, directional root growth away from salt was affected in their knockout mutants. Our approach exemplifies the use of simple phenotypes to unravel a complex signaling cascade and processes involved in initial responses to salt stress.

Results

Halotropism Is Directional Root Growth Response away from NaCl

The halotropism assay described by Galvan-Ampudia (Galvan-Ampudia et al., 2013) measures root growth and bending and can be used as an estimate for salt stress sensing and signaling in Arabidopsis by introducing a salt (NaCl) gradient (Figure 1A). In this study, we measured the root angle and increase in root length after gradient imposition (Figure 1B). The gradual increase in salt concentration allows the roots to adjust and mediate directional growth, toward areas of the plate with lower NaCl concentrations. Since the agar plates are placed in a slightly slanted position, at an angle of 70°, the roots exhibit a slight skewing to the right in control conditions (Figures 1C and S1; recorded as positive angles). The slight skewing observed on agar plates is due to the contact of the roots with the solid agar surface (Oliva and Dunand, 2007).

Figure 1

Halotropism Setup to Measure Arabidopsis Main Roots' Response to Salt

(A) Setup of halotropism assay. The assay performed on 0.5 MS agar medium measures root response to increasing NaCl concentrations.

(B–D) (B) Angle following gravity was represented as 0°, and the root angle and length post gradient were quantified. The boxplots represent the distribution of main root angle (C) and length (D) of 6-day-old Col-0 seedlings exposed to increasing concentrations of NaCl, LiCl, KCl, and sorbitol by introducing a gradient 24 h before (at 5 days). Results of a total number of 24 seedlings/condition were pooled from two biological replicates grown on 0.5 MS medium and analyzed. Statistical analysis of treatment versus control was done by two-way ANOVA with contrasts post hoc, where ***, **, and * represent p values <0.001, <0.01, and <0.05, respectively.

After 24 h treatment, directional growth away from NaCl concentrations was observed and recorded as negative root angles (Figure 1C). On the other hand, treatment with 20 mM LiCl resulted in root growth toward the LiCl medium, thus recorded as a positive angle (Figure 1C). The application of KCl, sorbitol, or 30 mM LiCl in the lower part of the medium did not induce a directional response (Figure 1C). Hence, a NaCl-specific root avoidance phenotype was observed. A reduction in root length was observed at both LiCl concentrations and at higher concentrations of NaCl, KCl, and sorbitol, whereas lower concentrations did not cause significant root length reduction, in comparison with seedlings grown on control plates (Figure 1D). Thus, root angle was used as a measure for the halotropic response, whereas increase in root length was used as a measure of general sensitivity toward both salt and osmotic stress.

At later time points (>48 h post stress) the root avoidance phenotype, denoted as negative angles, was also observed on KCl and sorbitol plates, although never to the same extent as root angles on NaCl plates (Figure S1). Seedlings on 20 mM LiCl-gradient plates retained positive root angles and did not avoid the increasing LiCl concentration throughout the experiment, but growth was completely inhibited on 30 mM LiCl at 48 h post stress (Figure S1). A decrease in root length was observed on both NaCl concentrations, 20 mM LiCl, and higher KCl and sorbitol concentrations at later time points (Figure S1).

Natural Variation in Root Angle Was Observed among Arabidopsis Accessions

A collection of 333 Arabidopsis accessions was grown and analyzed in six different experimental batches with internal controls (Table S1) consisting of the three accessions, Can-0, Col-0, and C24. No batch effect was observed (Figure S2), suggesting that the individual experimental batches could be combined. Accessions displayed extensive variation in main root angle values from 0° to 45° and from −25° to +25° on control and salt (NaCl) gradient plates, respectively (Figure 2A). Main root lengths varied between 0.2 and 0.8 cm and between 0.2 and 0.6 cm on control and salt gradient plates, respectively (Figure 2B), all at 24 h after medium imposition. Positive angles present accessions with roots skewing to the right, whereas those avoiding the salt skewed to the left, displaying negative angles (Figures 2 and S3).

Figure 2

Natural Variation of 333 Arabidopsis Accessions at 24 h

(A and B) Main root angle (A) and length (B) histograms indicating the distribution of 6day-old accessions at 24 h post gradient introduction.

(C) No correlation was observed between angle and length responses at 24 h post gradient introduction. Internal controls Can-0, Col-0, and C24 are indicated in the graph.

(D) The distribution of accessions based on angle independent of time points. New medium (control or salt) is denoted on the right side of the angle graphs, and root avoidance is clearly observed as negative root angles on the left side of the graph. A total number of 18 seedlings/accession/condition were quantified.

Natural variation in angle and length among the accessions was also observed at later time points, 48, 72, and 96 h post medium imposition (Figure S3). The root growth per day increased in control conditions (Figure S3), indicating an exponential increase in root growth as expected. In the period from 48 to 96 h on medium containing salt, roots started to grow again slowly, possibly due to the plant acclimation on salt, after recovery from a quiescent phase (Geng et al., 2013, Julkowska and Testerink, 2015). To determine if there was a correlation between root angle and length at any of the time points, response angle and length response were calculated as Root Angle CONTROL – Root Angle SALT and Length SALT/Length CONTROL, respectively. No correlation was observed between the root traits, showing that the halotropic response is independent of growth rate (Figures 2C and S4).

Some accessions had a naturally larger positive angle on control and responded slower to the salt gradient; hence, the root angle independent of time points was conceived as an alternative output for responsiveness to salt. Root angle on salt gradient plates independent of time points is basically a collection of the negative root angle data of accessions (Figure 2D) whenever the first negative angle was recorded and regardless of the time point post salt stress. In cases in which the root angle on salt of an accession remained positive, i.e., a non-avoidance phenotype, the root angle at 96 h post salt stress (the last time point of the experiment) was used as input for GWAS.

GWAS Identified Significant SNPs Associated with Halotropism

Individual phenotypic values and averages of the root traits of 333 Arabidopsis accessions were used as input data for GWAS and analyzed using a scan_GLS algorithm with 250,000 SNPs (Kruijer et al., 2014). Significant associations were found with several traits scored after gradient imposition. SNPs with a LOD score >5.6 and minor allele frequency >0.05 were selected. Heritability was the other parameter used for selecting putative candidates. The root angle traits that associated with the significant SNPs all had heritability values >0.2 (Table S4). The largest variation in root angle and length among the accessions was observed at 24 and 72 h post medium introduction, respectively (Figures 2A and S3D), and a higher number of significant SNPs was identified at these time points (Figures 3A, 3B, and S5 and Table S2). Fine mapping of the selected loci based on whole-genome sequencing data of different Arabidopsis accessions and 4,000,000 SNPs (Alonso-Blanco et al., 2016) identified multiple additional SNPs associated with the main traits (Table S3). Here, the log10 (p value) threshold was determined at 4. Genes underlying the 10-kbp window of significant SNPs identified with the scan_GLS algorithm had varying functional annotations (Huala et al., 2001), including receptor kinases, ion-binding proteins, genes involved in signal transduction, and protein and lipid modification, and a number of them were unknown (Table S2). A 10-kbp genome window was selected based on the average linkage disequilibrium of Arabidopsis (Molenaar and Keurentjes, 2014).

Figure 3

SNPs Associated with Root Angle on Salt

GWAS identified three (A) and two (B) SNPs that associated significantly with root angle traits at 24 h post salt stress. Divergence plots of a 6-kbp (C) and 5-kbp (D) region surrounding SNP B and E, respectively, based on accession sequences derived from the 1001 Genomes Project. Purple graphs signified missing data, green graphs indicated deletions in accessions other than Col-0, blue graphs represented similarity with Col-0, and the ORFs are shown in the last graph. The significant SNPs are denoted with dotted lines.

GWAS Candidate Genes Associated with Early Root Halotropism

The focus of our study was to unravel Na+ sensing and signaling in Arabidopsis roots by characterizing genetic components required for early halotropic responses, i.e., genes induced by Na+ at 24 h after application of a salt gradient. Hence, we concentrated on assessing natural variation at 24 h post gradient angle on salt for further analysis. We identified five significant SNPs, with one SNP per locus, using the scan_GLS algorithm that associated with main root angle on salt gradient plates at 24 h post stress or main root angle independent of time points (Table 1). Three SNPs mapped to main root angle on salt at 24 h were located in chromosomes 1, 2, and 5 (Figure 3A), whereas two SNPs associated with the time-independent trait and were found in chromosomes 1 and 4 (Figure 3B). Three SNPs associated with root angle on control at 24 h (Figure S5A), whereas no significant SNP was identified for root length at 24 h post medium introduction (Figures S5B and S5C).

Table 1

List of Putative Genes Identified by GWAS on Halotropic Responses

Trait	Chr	SNP Position	-log10(p value)	Col-0 Allele Frequency	Gene(s) in Region Around SNP
Root angle on salt, independent of time points	1	9376229(D)	6.73*4.95	0.09	At1g27000: UnknownAt1g27020: Unknown
Root angle on salt, at 24-h time point	1	11688813(A)	5.71*5.39	0.44	At1g32380 PRS2At1g32390: transposonAt1g32410: VPS55
		11688870+	4.24	0.40
Root angle on salt, at 24-h time point	2	12895120(B)	6.48*4.72	0.92	At2g30220: GDSL lipaseAt2g30230: UnknownAt2g30240: CHX13At2g30250: WRKY25
Root angle on salt, independent of time points		12903945+	4.49	0.45
Root angle on salt, independent of time points	4	13087635(E)	5.91*4.26	0.98	At4g25670: Unknown (DOB1)At4g25680: putative thiol peptidaseAt4g25690: Unknown
Response angle to salt at 24-h time point			4.92	0.47
Root angle on salt, at 24-h time point	5	21690498+	4.04	0.37	At5g53440: UnknownAt5g53450: ORG1At5g53460: GLT1
		21691399(C)	5.78*5.72	0.30
		21693328+	4.51	0.07

The putative candidates were selected based on the LOD score and minor allele frequency (MAF) of the SNPs associated with root halotropic responses. The Chromosome (Chr), position of a SNP locus, and MAF are indicated, including genes within the 10-kbp window surrounding the SNP. The three characterized candidate genes are highlighted in bold. SNPs identified by the two GWAS mapping methods are underlined, additional SNPs from fine mapping are indicated with a cross (+), and log10(p-value) scores from the scan_GLS program are indicated with an asterisk (*). (A) to (E) represent SNPs indicated in the Manhattan plots in Figure 3.

Next, by fine mapping, all SNPs identified via the GLS_scan program were confirmed and additional SNPs were identified (Tables 1 and S3). The 10-kbp region upstream and downstream of the significant SNPs was used to identify putative genes involved in halotropism. This resulted in a total of 15 candidate genes (Table 1) for the five loci of interest. In some cases, multiple SNPs were mapped to one locus for a single trait or multiple traits associated with the same SNP (Table 1). Two closely located SNPs on chromosome 1 associated with root angle 24 h post salt gradient imposition. On chromosome 5, three closely located SNPs associated with the same trait. Two closely positioned SNPs were identified on chromosomes 2 and 4 that associated with root angle 24 h after gradient imposition and root angle independent of time points.

A SNP on Chromosome 2 that was mapped to root angle after 24 h salt treatment (Table 1) was located at the 3′ end of CHX13, whereas the other SNP was positioned within the coding region of WRKY25 (Figure 3C). The variation in the genomic region surrounding the identified SNPs was produced using the sequences of the HapMap Arabidopsis accessions and showed extensive natural variation across the coding sequence of CHX13 (Figure 3C), as well as in the first exon of WRKY25, yielding both genes as potential candidates responsible for natural variation in halotropism responses. A candidate gene on chromosome 4 was named DOB1 (double bending 1), since the gene associated with two root bending traits (Table 1). A SNP located in Chromosome 4 of the Arabidopsis genome (Table 1) was positioned in the coding sequence of DOB1 (Figure 3D). Sequence alignment indicated that the coding sequence and promoter region of DOB1 is highly conserved among accessions, although a very small portion of the upstream region did show some divergence (Figure 3D).

To select follow-up candidate genes from the list, homozygous knockout mutants (Table S5) of the 15 putative genes within the 10-kbp interval surrounding the selected SNPs were phenotyped in the halotropism assay. It should be noted that no transfer DNA insertional lines were available for At1g32390. Most knockout alleles of the putative genes showed a similar root angle phenotype as Col-0 on control and salt, except wrky25-2 and dob1-1, which both exhibited a reduced halotropism response (Figure S6A). A number of mutants also differed from Col-0 in root length in both control and salt conditions (Figures S6B and S6C) but no trend between root angle responses and root growth was observed (Figure S6), indicating no contribution of one to the other. Hence WRKY25 and DOB1 were selected as follow-up candidate genes. CHX13 is the gene next to WRKY25, coding for a cation-proton antiporter and a possible transporter of K+, Na+, or Li+, making it an interesting follow-up candidate despite the fact that its knockout mutant was not affected in the halotropic response under the conditions used (high K+ in normal 0.5 MS medium). Hence, we continued with three candidate genes WRKY25, CHX13, and DOB1.

CHX13 Is Required for Root Halotropism under Low K+ Availability Conditions

Since CHX13 was reported to be a high-affinity K+ transporter (Zhao et al., 2008) it was necessary to check its halotropic phenotype on low K+ medium. For this purpose, the halotropic response on Modified MS medium (Spalding et al., 1999) containing 100 μM KCl was first checked for Na+ specificity and for root response in general, compared with 0.5 MS medium using Col-0 seedlings. Modified MS medium (MMS) contains minimal amount of nutrients, including K+, and has higher amounts of Ca2+ compared with 0.5 MS (Table S8). The 0.5 MS medium contains 10 mM of K+ (Murashige and Skoog, 1962) and will be referred to here as high-K+ medium, whereas the MMS medium contained 100 μM K+, hence referred to as low-K+ medium. Salt gradients of NaCl, LiCl, KCl, and sorbitol were introduced, and control plates were also included (Figure S7). Seedlings grown on low-K+ medium had a similar angle response to the ionic and osmotic gradients (Figure S7A) as those grown on high-K+ medium (described in Figure 1). In terms of root growth, the sensitivity of Col-0 seedlings to the stress and growth rate was different on high- and low-K+ media, as expected (Figures S7B and S7C). Hence, the use of halotropism assays made with either low- or high-K+ medium both supplemented with a 200-mM NaCl gradient can be used to assess Na+-specific responses of Arabidopsis seedlings.

Two independent knockout alleles (Figure S8) of WRKY25 and CHX13 were each phenotyped in the halotropism assay of high-K+ medium (0.5 MS with 10 mM KCl) and low-K+ medium (MMS with 100 μM KCl), both using 200 mM NaCl to establish the gradient. Knockout mutants wrky25-1 and wrky25-2 exhibited weaker response angles on salt gradient plates compared with Col-0 wild-type (WT) at 24 h post stress, irrespective of the K+ levels in the media (Figure 4A). The observed root response angle of the mutants was caused by significantly different root angles on salt gradient plates in both high- and low-K+ media compared with their Col-0 background, since they all had a similar root angle as Col-0 on control plates (Figure S9A). No difference was observed between the mutants and Col-0 in main root length in control and salt conditions (Figure S9B), again iterating that root growth did not correlate with halotropic responses.

Figure 4

WRKY25 and CHX13 Are Required for Early Halotropic Responses in Arabidopsis Roots

The boxplots represent the distribution of main root halotropic responses of 6-day-old WRKY25 (A) and CHX13 (B) mutants grown on high- (0.5 MS with 10 mM K+) or low-K+ (MMS with 100μM K+) medium supplemented with a 200-mM NaCl gradient. The location of transfer DNA insertions in the genes are indicated above the graphs. Response angle was calculated as: Root Angle CONTROL – Root Angle SALT. A total of 24 seedlings/genotype/condition and two biological replicates were quantified at 24 h post stress. Relative expression of WRKY25 (C) and CHX13 (D) transcripts in 6day-old Col-0 roots grown on high- (0.5 MS with 10 mM K+), sufficient- (MMS with 200 μM K+), or low-K+ (MMS with 100 μM K+) medium supplemented with 100 or 200 mM NaCl gradient. Data are represented as mean ± SEM. Roots were harvested at the 24-h time point, and at least 80 seedlings/condition/RNA sample from three biological replicates were used. Statistics was by two-way ANOVA with Tukey post hoc, where different letters represent p values < 0.05.

For CHX13, the homozygous knockout alleles chx13-1 and chx13-2 exhibited a similar response angle as Col-0 on high-K+ medium, but when seedlings were grown on low-K+ medium, both chx13-1 and chx13-2 exhibited significantly weaker response angles than Col-0 at 24 h post stress (Figure 4B). The main root angle of both knockout alleles did not differ from Col-0 on control plates of high- or low-K+ medium (Figure S9C). This indicates that the response angle on low-K+ medium was due to a change in root angle on salt gradient plates (Figure S9C). Root growth of both chx13 knockout alleles did not significantly differ from Col-0 on control and salt conditions (Figure S9D). These results indicate that CHX13 is involved in early halotropic responses, but only under limiting K+ conditions.

Taken together, WRKY25 is required for halotropic responses, whereas CHX13 contributes to root halotropism only under limiting K+ conditions.

WRKY25 and CHX13 Are Upregulated in the Root in Response to Salt Stress

To investigate whether WRKY25 and CHX13 are transcriptionally regulated during our salt stress experiments, Arabidopsis Col-0 seedlings were grown on media with different K+ levels: in 0.5 MS containing 10 mM K+ (high K+), MMS with 200 μM K+ (sufficient K+), or MMS with 100 μM K+ (low K+). Salt treatments were applied as a gradient of 100 mM NaCl (mild salt stress) or 200 mM NaCl (high salt stress) for 24 h, and control plates (medium without salt) were also included. The expression of the three candidate genes in the roots was examined. We observed upregulation of WRKY25 transcripts in the root under both mild and high salt conditions on sufficient and low-K+ medium but not in high-K+ MS medium (Figure 4C), whereas the expression of WRKY25 was unaltered by different K+ levels in the absence of salt (Figure 4C). This suggests that WRKY25 might play a role in halotropism through transcriptional upregulation and its transcriptional induction might rely on an imbalance between Na+ and K+. Expression of CHX13 on the other hand was responsive to K+ levels even under control conditions, with the highest CHX13 transcript levels observed under low-K+ conditions (Figure 4D), supporting its role in transporting K+ with high-affinity characteristics (Zhao et al., 2008). Interestingly, K+-dependent upregulation was even more pronounced under salt stress conditions (Figure 4D). This suggests that CHX13 has a specific role in halotropic responses linked to the interplay between Na+ and K+. Typically, salt stress reduces the availability of K+ in plant tissue, possibly replicating a K+ limiting condition where the high-affinity K+ transporter CHX13 is involved. Thus, CHX13 transcript levels increased in K+-limiting conditions and both WRKY25 and CHX13 expression were upregulated early in Arabidopsis roots during salinity stress.

CHX13 Is Required for Ion Accumulation during Salt Stress

To link the initial salt stress responses with long-term salt tolerance, we decided to determine if mutants with root angle phenotypes would be affected in ion accumulation in the shoot tissues of older plants. Here, Arabidopsis plants (WT Col-0 and mutants) were grown hydroponically with salt treatment up to 100 mM NaCl for 1 week. The Na+ and K+ content in the shoot were measured in 4-week-old plants. In control conditions, chx13 knockout mutants had similar Na+ and about 6% less K+ content in the shoot, compared with Col-0 WT (Figures 5A and 5B).

Figure 5

CHX13 Is Required for Na+ and K+ Accumulation during Salt Stress

(A and B) Na+ content (A) and K+ content (B) in the shoot of Col-0 plants and chx13 knockout mutants in control and salt conditions.

(C) Fold change (Treatment/Salt) of shoot Na+/K+ ratio.

(D) The shoot dry weight and main root length in response to salt. Response was calculated as: Treatment/Control. Arabidopsis seedlings (Col-0, chx13-1, and chx13-2) were hydroponically grown for 4 weeks (1 week stress of 100 mM final NaCl concentration) on Hoagland medium with sufficient K+ (200 μM K+) and harvested. Graphs are quantified data from nine replicates/genotype/condition in one biological experiment.

Data are represented as mean ± SEM, and statistics was by two-way ANOVA with Tukey post hoc, where different letters represent p values < 0.05.

Under salt stress conditions, chx13 mutants accumulated significantly less Na+ (Figure 5A) and had higher shoot K+ content than Col-0 (Figure 5B), indicating that CHX13 may function in maintaining the balance between Na+ and K+ ions in the shoot during salt stress. Consequently, a lower Na+/K+ ratio was observed in the chx13 lines in response to salt stress (Figures 5C and S10A), suggesting improved maintenance of ion balance in the mutant lines. Both knockout alleles of CHX13 had lower shoot biomass and longer main roots than Col-0 WT in control and salt conditions (Figure S10B), but the biomass reduction in response to salt stress was smaller in the mutant lines compared with Col-0 (Figure 5D). This suggests that the maintenance of ion balance between Na+ and K+ ions might contribute to the improved salinity tolerance of chx13 mutant lines, expressed as reduced salt stress sensitivity in shoot dry weight and root length.

DOB1 Is Required for Root Halotropism and Upregulated by Salt Stress

The two homozygous knockout mutants dob1-1 and dob1-2 (Figure S8) significantly differed from Col-0 WT in their response angle on both high- and low-K+ media at 24 h post stress (Figure 6A), indicating that the main root halotropic response influenced by DOB1 occurs independently of K+ levels. The main root angle of the mutants was similar to Col-0 on control plates with high and low K+, whereas both DOB1 knockout alleles differed significantly from Col-0 on salt plates (Figure S9E), indicating that the difference in response angle was due to changes in root angle on salt gradient plates. Hence, DOB1 is required for halotropic responses. Both dob1-1 and dob1-2 had significantly shorter roots than Col-0 on control and salt conditions (Figure S9F), again iterating that root length did not contribute to observed root angle phenotype. No significant change in DOB1 expression was observed in response to varying K+ levels in the media (Figure 6B). However, both a mild and high salt gradient in the media containing sufficient and low K+, but not high K+, caused a significant increase in DOB1 transcripts (Figure 6B), suggesting that DOB1 could play a role in halotropic response through transcriptional upregulation.

Figure 6

DOB1 Is Involved in Root Halotropic Responses

(A) The boxplot represents the distribution of the main root halotropic responses of 6-day-old DOB1 (A) mutants grown on high- (0.5 MS with 10 mM K+) or low-K+ (MMS with 100 μM K+) medium supplemented with a 200-mM NaCl gradient for 24 h. The location of transfer DNA insertions in the gene is indicated above the graph. A total of 24 seedlings/genotype/condition and two biological replicates were quantified.

(B–D) (B) DOB1 transcripts in 6-day-old Col-0 roots grown on high- (0.5 MS with 10 mM K+), sufficient- (MMS with 200 μM K+) or low-K+ (MMS with 100 μM K+) medium supplemented with a 100- or 200-mM NaCl gradient. At least 80 seedlings/condition/RNA sample and three biological replicates were used. Data are represented as mean ± SEM. The boxplots represent the distribution of root halotropic (C) and length responses (D) of 6-day-old accessions with differential DOB1 expression grown on 0.5 MS (with 10 mM K+) medium supplemented with a 200-mM NaCl gradient. In total, 24 seedlings/genotype/condition from two biological replicates were quantified at the 24-h time point, and the figure represents one of the experiments. Statistics was by two-way ANOVA with Tukey post hoc, where different letters represent p values < 0.05.

Arabidopsis accessions with documented contrasting expression of DOB1 were phenotyped for their halotropic responses on 0.5 MS medium (high-K+ medium) only supplemented with a 200-mM NaCl gradient for 24 h. Public expression data indicated that C24 and Is-0 are accessions with high expression of DOB1, whereas Est and Sf-2e have low expression of DOB1 (Lempe et al., 2005, Winter et al., 2007). In our halotropism assay, C24 had a similar response angle as Col-0, whereas Is-0 displayed a much stronger response angle than Col-0 (Figure 6C). Both Est and Sf-2e had significantly weaker response angles than Col-0 (Figure 6C). Sf-2e had a similar root angle as Col-0 on control plates, whereas the other three accessions had more positive root angles on control plates than Col-0 (Figure S9G). On salt gradient plates, C24 and Is-0 exhibited root avoidance phenotypes similar to Col-0, whereas the accessions with low DOB1 expression, Sf-2e and Est, had root angles of −5° and +24°, respectively, differing from Col-0 (Figure S9G). Thus both root angle on control and salt (Figure S9G) contributed to the observed response angle (Figure 6C), and a clear difference was observed between groups of accessions with high and low DOB1 expression. In terms of root length, no specific trend was observed for the accessions (Figures 6D and S9H) that could link root growth to angle response (Figures 6C and 6D). Taken together, lower levels of DOB1 correlated with a reduced salt avoidance phenotype of roots at 24 h post stress, whereas higher levels of DOB1 correlated with root avoidance of salt gradient, in this subset of accessions. This was consistent with the observed phenotype of DOB1 knockout alleles (Figures 6A and S9E), indicating that DOB1 is required for early root halotropic responses.

DOB1 Is Involved in Na+/K+ Accumulation during Salt Stress

To investigate a possible correlation between the observed root angle and length phenotypes with Na+/K+ accumulation in the shoot, Na+ and K+ content were measured in the dob1 knockout mutants. A slight difference was observed in control conditions between the knockout mutants and Col-0 (Figures 7A and 7B), similar to previous observations for the chx13 mutants (Figures 5A and 5B). In response to salt, both dob1 mutant lines amassed lower amounts of Na+ (Figure 7A) and a slightly higher K+ content (Figure 7B) in the shoot compared with Col-0 WT, resulting in the knockout mutants exhibiting a reduced Na+/K+ ratio in response to salt (Figures 7C and S11A). Hence, DOB1 plays a role in Na+/K+ homeostasis during salt stress. The dob1 mutants were smaller than Col-0 WT in control conditions (Figure S11B). In response to salt, these mutant lines had similar shoot biomass but smaller roots lengths compared with Col-0 (Figure S11B). When expressed as a response to salt (Figure 7D) the knockout alleles had decreased salt sensitivity in the shoot, which may contribute to improved tolerance to salt stress. The reduced root growth in response to salt (Figure 7D) may also influence ion accumulation in these tissues.

Figure 7

DOB1 Also Plays a Role in Na+ and K+ Accumulation during Salt Stress

(A and B) Na+ content (A) and K+ content (B) in the shoot of dob1 knockout mutants in control and salt conditions.

(C) Fold change of shoot Na+/K+ ratio in the mutants.

(D) The shoot dry weight and main root length in response to salt. Arabidopsis seedlings were hydroponically grown for 4 weeks (1 week stress of 100 mM final NaCl concentration) on Hoagland medium with sufficient K+ (200 μM K+) and harvested. Graphs are quantified data from nine replicates/genotype/condition in one biological experiment.

Data are represented as mean ± SEM, and statistics was by two-way ANOVA with Tukey post hoc, where different letters represent p values < 0.05.

Discussion

A compelling way in which plants can adapt to their environment is by adjusting their growth away or toward a stimulus, and these directional growth responses are termed tropisms. Shoots typically grow toward sunlight, whereas root growth is directed by gravity, water, and nutrient availability (Feng et al., 2016, Sato et al., 2015). Directing root growth away from high concentrations of salt is called halotropism (Galvan-Ampudia et al., 2013), and this response can easily be observed in Arabidopsis seedlings grown on salt gradient agar plates. Main root angle is a quantitative phenotype used to describe the salt-induced directional root growth away from salinity (Figure 1). The salt gradient or halotropism assay shows a Na+-specific root avoidance phenotype at 24 h post stress. This phenotype is reproducible and quantifies root angle as an output of salt sensing or signaling (Figure 1). Our experiments employed a gradient assay, allowing gradual root exposure to increasing NaCl concentrations.

Lithium and sodium are alkali metals inducing ionic toxicity, although the former reduces root growth at much lower concentrations (Figures 1D and S1). In our assay, Arabidopsis seedlings do not display root avoidance but grow toward the LiCl-gradient, except when growth was inhibited (Figure S1). Although a number of genes are induced by both Na+ and Li+ stresses (Yokoi et al., 2002), these two do not seem to have an overlapping signaling mechanism, and a lithium-tolerant mutant cat2 is actually hypersensitive to Na+ (Bueso et al., 2007). Potassium deficiency rather than excessiveness is the typically observed stress in plants, hence the widespread application of NPK fertilizers (Anschütz et al., 2014, Ashley et al., 2006, Shabala and Cuin, 2008). Our experiments showed reduced root growth on addition of 200 mM of KCl that was similar in response to an equi-osmolar concentration of sorbitol (Figures 1D and S1), indicating an osmotic but not an additional ionic stress response. Hydrotropism assays with 400–812 mM of sorbitol previously reported a directional root change (Dietrich et al., 2017) at an early (12 h post stress) time point, which was not observed in our experiments (Figure 1C). Several differences can be noted between the hydrotropism assay reported (Dietrich et al., 2017) and our setup, including the root distance from new medium imposition and sorbitol concentration. We hypothesize that hydrotropism occurs at much higher osmotic values than the halotropism response, which occurs at lower salt concentrations.

Our study focused on unraveling and characterizing genetic components contributing to early halotropic responses. GWAS has been successfully used in recent times to identify important genetic components for acclimation strategies employed by plants to cope with salt stress (Julkowska et al., 2017, Julkowska et al., 2014, Kawa et al., 2016). Since little is known about Na+-specific root growth away from salt except its dependence on auxin transport and re-distribution (van den Berg et al., 2016, Galvan-Ampudia et al., 2013, Sun et al., 2015), it was important to identify other genetic components that are required for early signaling in response to salt stress. The halotropism assay was used to screen a HapMap population of 333 Arabidopsis accessions to study natural variation in root halotropic responses that was subsequently used for GWAS. WRKY25, CHX13, and DOB1 were selected as follow-up candidate genes from an initial selection of 15 candidate genes (Table 1). These three genes were linked to SNPs that associated with main root angle and response angle to salt at 24 h after medium imposition (Table 1 and Figure 3).

WRKYs are plant-specific transcription factors that play a role in gene regulation and localize to the nucleus, like other transcription factors. All family members (>50) contain the highly conserved WRKY domain, are randomly distributed in the Arabidopsis genome, and directly target genes that have “W-boxes” in their promoter region, including other WRKYs (Eulgem et al., 2000). WRKY25 is upregulated during salt stress, and knockout mutants exhibited reduced halotropic responses (Figures 4A and 4C), indicating that WRKY25 plays a role in halotropism. WRKY25 has two WRKY domains and belongs to Group 1 of the WRKY family, which also includes WRKY33 (Eulgem et al., 2000). Both WRKYs are induced within 6 h in response to a number of abiotic stresses, but salt-induced WRKY25 expression is independent of abscisic acid (ABA) and salt overly sensitive signaling (Jiang and Deyholos, 2009, Li et al., 2009, Li et al., 2011).

A yeast two-hybrid screen identified WRKY25 as a direct interactor of MAP kinase substrate 1 (MKS1) (Andreasson et al., 2005). In vitro kinase assays indicated that the WRKY was phosphorylated by MAP kinase 4 (MPK4), and MPK4 also phosphorylates MKS1. MPK4 is a mitogen-activated protein (MAP) kinase required for the activation of jasmonate-dependent responses and the suppression of salicylic acid-dependent responses in planta during pathogen attack (Andreasson et al., 2005). Affinity purification followed by mass spectrometry identified another protein interactor of WRKY25, growth-regulating factor 3 (GRF3). GRF3 is a 14-3-3 isoform ψ protein required for main root growth under potassium and nitrogen starvation (Shin et al., 2010). Microarray analysis indicated that WRKY25 induces a wide range of genes involved in numerous physiological processes (Jiang and Deyholos, 2009). Hence, WRKY25 may contribute to root halotropism by directly targeting genes required for acclimation during salt stress.

Cation proton exchangers (CHXs) belong to the monovalent CPA2 (cation proton exchanger 2) family consisting of twenty-eight members. They are described as possible K+/H+ or Na+/H+ antiporters involved in maintaining intracellular pH and ion homeostasis (Sze and Chanroj, 2018, Sze et al., 2004). CHX13 localizes to the plasma membrane and was reported to be induced in the roots during K+-limiting conditions (Zhao et al., 2008), similar to our observations (Figure 4D). We find that CHX13 is upregulated during salt stress and is required for early halotropic responses in roots (Figures 4B and 4D). Potassium is an essential macro-nutrient, required for plant growth and development. Potassium deficiency is linked with the ionic component of salinity stress, since Na+ displaces K+ for cellular function (Ashley et al., 2006). The identification of a K+ transporter that is involved in root halotropism reinforces the importance of sustaining cytosolic Na+/K+ balance during increased salt stress, even during the early stages of salt stress exposure.

DOB1 is an unknown gene with a possible link to ABA signaling through its predicted interaction (Stark et al., 2006) with MYB49 (Lumba et al., 2014). It is upregulated in the roots during salt stress at sufficient- and low-K+ conditions (Figure 6B), and reducing the expression of DOB1 reduced halotropic responses in Arabidopsis roots (Figures 6A and 6C). Hence, it plays a role in main root growth away from higher salt concentrations. Although DOB1 transcript was not upregulated by salt on 0.5 MS medium at 24 h (Figure 6B), information from public expression data indicates that this gene is upregulated only at earlier time points, specifically before 24 h (Dinneny et al., 2008, Kilian et al., 2007, Winter et al., 2007). This earlier transcriptional increase may still have an impact on the halotropic responses observed at 24 h post stress.

The mutant lines of both CHX13 and DOB1 were able to better maintain their Na+/K+ ratio in the shoot than Col-0 WT, after long-term salt exposure (Figures 5C and 7C), indicating that both genes function in ion accumulation and maintaining ion homeostasis during salt stress. Previous observations showed that K+ transport mediated by CHX13 in yeast was inhibited in the presence of Na+ (Zhao et al., 2008). Yet, it is not known whether Na+ would compete with K+ for transport in this case. How the observed lower Na+ accumulation in chx13 and dob1 mutants in salt conditions (Figures 5A and 7A) relates to their reduced halotropic responses (Figures 4A and 6A) is still unknown. Both responses were observed at very different time scales, and the consequence of early salt stress responses such as halotropism and salt stress tolerance in adult plants remains to be established. The significantly reduced shoot Na+ content of chx13 mutants (Figure 5A) would suggest that CHX13 protein may function in either Na uptake from soil by the roots or Na+ transport to the shoot during salt stress. In support of this idea, the antiporter CHX21, which is expressed in endodermal root tissues, functions in xylem loading of Na+, resulting in Na+ accumulation in the leaves (Hall et al., 2006). Although CHX13 plays a role in root halotropism and salt stress tolerance, it is not unique in K+ transport during limiting conditions. High-affinity K+ transporter 5 (HAK5) and CHX17 are major transporters involved in K+ transport during starvation (Ashley et al., 2006, Gierth et al., 2005). CHX17 is a well-characterized high-affinity transporter mediating K+ homeostasis in the roots (Cellier et al., 2004). CHX14 is a close relative of CHX13 (Sze et al., 2004) and is involved in K+ transport and re-distribution in the shoot and roots but functions in elevated K+ conditions (Zhao et al., 2015).

The Plant Transcription Factor database, Plant TFDB (Jin et al., 2017), predicts the ARF family as one of the top putative transcription factors binding to the promoter regions of DOB1 and CHX13. Auxin response factors (ARFs) regulate other genes by repressing or promoting their activity and requires another transcription factor Aux/IAA repressors to confer auxin response (Guilfoyle and Hagen, 2007). Thus, regulation of transcription could provide a link between the players identified here and the observed auxin dependence of the root halotropic response. A number of WRKYs, not including WRKY25, were also predicted to bind the promoter of DOB1 in the Plant Transcription Factor database, whereas only WRKY17 was predicted to bind the promoter region of CHX13 (Jin et al., 2017). Although the long-term effects of the halotropism response remain elusive at this stage, the halotropism assay provides an efficient readout to characterize natural variation in salinity response and was used here to identify additional genetic components required for early salt-specific halotropic responses in Arabidopsis roots, which also affect salt stress acclimation.

Limitations of the Study

Although we discovered and characterized salt-specific genetic components required for halotropism, shoot growth, and ion accumulation in Arabidopsis, a direct link between maintaining shoot Na+/K+ homeostasis and the root halotropic responses remains unknown. Additional experiments will be required to assess a possible role of the CHX13 protein in K+/Na+ transport during salt stress. The long-term impact of halotropism, in terms of plant survival and crop yield in saline soils, also remains elusive at this stage.

Methods

All methods can be found in the accompanying Transparent Methods supplemental file.