Natural variation among Arabidopsis thaliana accessions in tolerance to high magnesium supply

Abstract

High magnesium (Mg²⁺) in some extreme serpentine soils or semi-arid regions is an important factor affecting crop growth and development. Specific loci that form the genetic framework underlying high Mg²⁺ homeostasis, however, are not well understood. By using GWA mapping on 388 accessions of Arabidopsis thaliana selected from a worldwide collection and genotyped at approximately 250,00 SNPs, we successfully identified 109 and 74 putative genetic regions associated in nutrient traits under normal (1,000 µM) and high Mg²⁺ (10,000 µM), respectively. Above 90% SNPs associated with nutrients including Mg²⁺ and only two SNPs shared between normal and high Mg²⁺. A single strong peak of SNPs associated with Ca concentration corresponding to candidate gene At1g60420 ARABIDOPSIS NUCLEOREDOXIN (AtNRX1) under high Mg²⁺ was further determined. Compared with wildtype, mutants of Atnrx1-1 and Atnrx1-2 supplied with high Mg²⁺ had higher Ca concentrations in the plant, and higher cytosolic Ca²⁺ concentrations during root elongation, as well as higher fresh weight and lateral-root number. This suggests that AtNRX1 was a critical gene negatively regulating Ca uptake under high Mg²⁺ conditions. The discovery could help to breed/select crops that can adapt to high-Mg²⁺ soils such as serpentine soils (high ratio of Mg²⁺: Ca²⁺) or Mars soil with high levels of magnesium sulfate.

Introduction

Magnesium (Mg) is the 8^th most abundant mineral element on earth and the fourth abundant mineral element in plants following nitrogen (N), potassium (K) and calcium (Ca)¹. Magnesium in soils originates from source rock material containing various types of silicates and carbonates. In extreme serpentine (high magnesium: calcium) soils² and in semi-arid regions, where water stress conditions³ can lead to Mg accumulation. In addition, Mars regolith (unconsolidated surface material) is a potential medium for plant growth in bioregenerative life support systems. However, analyses by the Mars Exploration Rover Landers at Meridiani Planum and Gusev Crater have also suggested there are particularly high levels of magnesium sulfate minerals in outcrops and soils^4–6.

Although plants rely on a sufficient supply of Mg²⁺ for normal growth and development, excessive Mg²⁺ accumulation often causes toxicity to plant cells. Plants grown in high-Mg²⁺ soils like serpentine or Mars soils may alleviate Mg toxicity by limiting internal Mg accumulation, Mg excretion from leaves and/or increasing plant tolerance to high Mg²⁺ concentrations via altering leaf size, sclerophylls, stature and root systems^7,8. Previous reciprocal transplant experiments between serpentine populations (or species) and their spatially adjacent populations (or sister species) growing on serpentine soils demonstrated unequivocally that adaptation to serpentine soils had a genetic basis^9–12. Several quantitative trait loci that control contrasting adaptive traits in serpentine-tolerant Microseris douglasii and serpentine-intolerant M. bigelovii have also been identified¹³. A high degree of polymorphism has been reported among plant species in terms of growth tolerance to serpentine soils^14–16.

This present study employed genome-wide association studies (GWAS), an approach that has been applied successfully to identify many different traits ranging from genetically simple traits to more complex features¹⁷, to identify candidate genes likely to be involved in adaptation to high Mg²⁺, based on 388 accessions of Arabidopsis thaliana selected from a worldwide collection. Arabidopsis thaliana has a broad geographical distribution and consequently is subject to varying nutritional environments, which makes it a useful model species for studying nutrient homeostasis^18,19 in a wide variety of environments²⁰. Importantly, more than 1300 distinct accessions have been genotyped²¹ and to date more than 1000 inbred lines have been ‘fully sequenced’. Meanwhile, automated phenotyping platforms have been developed^22–24, allowing for precise and high-throughput measurements of plant growth. These developments enhance our ability to map causal genetic polymorphisms through GWAS. Moreover, high Mg²⁺ decreased the growth of Arabidopsis including biomass production, root hair development and the absorption of other nutrients^25–28. To further identify the likely candidate genes that are involved in adaptation to high-Mg²⁺ soils, we used a chemically-defined nutrient solution that contained 10,000 μM MgSO₄ of maximum authentic high Mg²⁺ soils to perform a viability screen on a large collection of wild-type accessions of Arabidopsis thaliana. This study aimed to (1) investigate natural variations in growth response to high Mg²⁺, and (2) identify novel genes tolerant to Mg²⁺ excess.

Materials and Methods

Plant material and growth conditions

Seeds of 388 accessions with accessions CS76636, CS76427, CS78885 and CS22660 and two T-DNA insertion lines SALK_100357C and SALK_113401C were derived from the Arabidopsis Biological Resources Center stock center (http://nasc.nott.ac.uk). The accessions were selected without any a priori consideration of their natural habitat, including geographical distribution or precipitation levels, and were collected from the wild and available in the stock center or soon-to-be-released collections. Most of these accessions were originated from Europe including Sweden, Germany, France, Czech Republic and United Kingdom, and from North America and Middle Asia, representing the sampling strategy of the 1,307 worldwide accessions²¹. This was consistent with the distribution of the 1,307 Arabidopsis lines collected in RegPanel.

Arabidopsis cultivation

The 388 accessions of Arabidopsis thaliana were grown under 2 Mg²⁺ treatments, which were achieved by altering the concentration of MgSO₄ in the basal medium. To minimise maternal effects prior to phenotyping, natural accessions were grown for one generation during 2015 under controlled greenhouse conditions at Zhejiang University (N30°18′25, E120°04′54). For surface sterilization, seeds of various accessions were placed for 1 h in open 200-μL PCR tubes in a sealed box producing chlorine gas generated from 13 mL of 10% sodium hypochlorite and 350 μL of 37% hydrochloric acid. Sterile seeds were then put on the surface of 30 mL agar media, containing 1.2% (w/v) agar and 0.6% (w/v) sucrose (A-1296; Sigma-Aldrich; http://www.sigmaaldrich.com) in 10 × 10-cm² plates with grid schematic engraved below the plate. Plates were positioned in racks and oriented in a vertical position, and were kept at 4 °C for 48 h in the dark for seed stratification. Thereafter, the racks containing the plates were transferred to a growth chamber under a 10-h light/14-h dark photoperiod at constant temperature of 22 °C, 60% relative humidity and light intensity of 120 µmol photons m⁻² s⁻¹. Twelve seedlings were grown in each plate and each treatment received at least four independent replicates. The racks were removed to the image acquisition room once per day and then immediately returned to the growth chamber. Throughout the experiments, the plate position within the box and box position in the growth chamber were re-randomized daily. For assays on agar plates, studies were performed on 8-d-old plants; that is, at an early stage of their stability and homogeneity growth phase. Moreover, many previous publications adopted 6- to 8-d-old Arabidopsis seedlings under stresses for determining Arabidopsis morphology, physiology, and development as well as responses to nutrient stresses^29–31.

Mg²⁺ Treatments

The basal medium (Normal Mg²⁺, N), which was used as control, contained (µM) 1500 KNO₃, 500 NaH₂PO₄, 1000 CaCl₂, 250 (NH₄)₂SO₄, 1000 MgSO₄, 25 Fe-EDTA, 10 H₃BO₃, 0.5 MnSO₄, 0.5 ZnSO₄, 0.1 CuSO₄ and 0.1 (NH₄)₆Mo₇O₂₄. The normal and high Mg²⁺ treatments contained 1,000, and 10,000 µM MgSO₄, respectively, which was in accordance with Mg²⁺ concentrations in soil solutions²⁷. 10,000 μM Mg²⁺was selected in the present study for the quantitative analysis of the natural variation of Arabidopsis in response to high Mg²⁺ stress because this concentration had an adverse effect on growth without toxic symptoms of most natural accessions^27,32. Besides, the concentrations of Mg²⁺ in the control were 1000 µM, that has been adopted for Arabidopsis growth by many plant biologists^33–35. Though the concentration of SO₄²⁻ increased 10 times comparing with that in the normal medium, SO₄²⁻ had hardly affected morphogenesis of Arabidopsis^27,36. The pH of the growing media was adjusted to pH 5.8 with MES (N-morpholino) ethane-sulphonic acid)-KOH buffer before autoclaving.

Phenotype analysis

Each line had four biological replicates for each treatment. Seeds that had not germinated at day 6 were discarded from further analysis, resulting in approximately 3–4 seedlings analyzed per genotype per treatment. The 21 traits under each treatment were classified into 2 categories: 7 morphological traits and 14 nutrient traits. Seedlings were photographed with a high-resolution digital camera (Sony RX100, Japan) daily for determination of root and shoot growth. Root or shoot biomass, and the number of days from seeding to emergence (>50% seedlings having the first radicle or cotyledon), were recorded. Meanwhile, photographs after 8 days of treatment were analyzed and quantified for phenotype using the public domain image analysis program Image J version 1.43 (http://rsb.info.nih.gov/ij/)²⁷. The length of primary roots, rosette diameter and epicotyl were determined across the median seedling using Image J. Lateral-root number was determined by counting the number of true roots (>1 mm long lateral root primordia) per primary root. For each condition, an independent sample accession consisted of four plants at the same growth stage, and the time of sampling was the end of the light period of day 8.

Analyses of mineral homeostasis

After 8-d growth at various Mg²⁺ concentrations, plants were harvested. All fully-expanded seedlings without lesions were collected, and weighed. The results were the average value across all available replicates. Seedlings were washed thoroughly with ultrapure water and dried in an oven at 75 °C for 24 h. The dried root and shoot samples were then wet-digested in the concentrated HNO₃/H₂O₂ at 90, 120 and 140 °C for 2 h, respectively, until there was no brown fume, and then further digested at 180 °C until the digest became clear. Concentrations of potassium (K), calcium (Ca), Magnesium (Mg), sulfur (S), iron (Fe), manganese (Mn) and sodium (Na) in the digests were analyzed by ICP-MS (Inductively coupled plasma mass spectrometer, Agilent 7500a, USA), the contents per plant were calculated. Mineral analysis was done in the whole-plant.

Fluo 4-AM loading and confocal imaging of cytosolic Ca²⁺ concentration in root

The Ca²⁺-sensitive fluorescent dye 1-[2-amino-5-(2,7-difluoro-6-hydroxy-3-oxo-9-xanthenyl)phenoxy] -2-(2-amino-5-methylphenoxy)ethane-N,N,N′,N′-tetraacetic acid, pentaacetoxymethyl ester (Fluo-4/AM) ester was used to estimate cytosolic Ca²⁺ concentration in the roots. The Fluo-4/AM ester was loaded into the root at 37 °C in the dark at a final concentration of 5 μM Fluo 4-AM dye and Pluronic F-127 (0.05%) in the Hank’s Balanced Salt Solution (HBSS) solution. After 1-h incubation, the roots were washed with the standard HBSS medium three times and left at 25 °C for 30 min. The samples were mounted and photographed with a Zeiss LSM 780 (Zeiss Co., Germany). Fluorescence was detected using 488 nm excitation and 505–545 nm band pass filter. Roots including root hairs were placed on the stage of a Zeiss Axiovert inverted microscope attached to a LSM780 laser scanning confocal microscope, and imaged using a Zeiss 20×, N.A., wet objective. Each frame represents a 7-s scan of the laser.

Genome-Wide Association (GWA) mapping, SNP selection, and statistics

The GWA mapping was performed on the values of epicotyl length, rosette width, primary root length, and lateral root number of the median seedling of each accession. For traits of and nutrients, means values of 4 biological replicates were used. We linked 21 traits under normal or high Mg²⁺ (Supporting Information Table S1) to published genomic data on accessions from a 250 K SNP chip. The GWA analysis was performed in the GWAPP web (http://gwas.gmi.oeaw.ac.at/) interface using the mixed model algorithm method³⁷. A kinship matrix was generated using the identity in state of SNPs between each pair of accessions³⁸. The P-value above bias due to population stratification was evaluated with Q–Q plots. Candidate loci were selected based on the minor allele frequency, LOD score and trait-associated SNPs and their position in the genome to pinpoint candidate genes within 5 kb up- and down-stream of the identified SNP (Supporting Information Table S2). The putative candidate genes corresponding to each SNP are listed in Supporting Information Table S3. For each of the candidate genes, the annotations were retrieved from TAIR10 (Arabidopsis.org).

Extraction of total RNA and quantitative PCR

Samples were collected from 8-day-old Arabidopsis after grown at normal and high Mg²⁺ treatments. For each condition, four samples were taken on independent seedling materials from eight different plants at the same growth stage and the time of sampling was the end of the light period of day 8. Total DNA was extracted by Plant DNA Isolation Reagent (Takara) from about 80 mg of fresh seedling tissues of four independent biological replicates. Two homozygous T-DNA insertion lines (SALK_100357C, Atnrx1-1) and (SALK_113401C, Atnrx1-2) was conducted by PCR with gene-specific primers SALK_100357C LP: 5′-AACCCAACCATCTTTGGACTC-3′; SALK_100357C RP: 5′-TCAAGACTTCAAGACCAAGCC-3′ and T-DNA border primer LBb1.3:5′-ATTTTGCCGATTTCGGAAC-3′; SALK_113401C, LP: 5′-ATCCACTTTTGTCGTTGAACG-3′; SALK_113401C RP: 5′-CGATCGCAACTTCTTCTGATC-3′ and T-DNA border primer LBb1.3:5′-ATTTTGCCGATTTCGGAAC-3′; Total RNA was extracted by RNAisoPlus (Takara, Otsu, Shiga, Japan) from about 70 mg of fresh seedling tissues of four independent biological replicates. All RNA samples were checked for DNA contamination before cDNA synthesis. cDNA was synthesized, and possible residual genomic DNA contamination was verified as described in our previous study³⁹. The mRNA levels of AT1G60420 in two lines of SALK_100357C and SALK_113401C were detected by the SYBR Green RT-PCR kit (Takara) with following pairs of gene-specific primers: AT1G60420, fw: 5′- TTCTCGGCCCTGATGGAAAAACC-3′, rv: 5′- GCTTCATTCCGCTCCTTTATCTGCT-3′ A pair of housekeeping gene of UBQ10 (fw: 5′-GGTTCGTACCTTTGTCCAAGCA-3′, rv: 5′-CCTTCGTTAAACCAAGCTCAGTATC-3′) was used for a control PCR. Melting curve analysis and gel electrophoresis of the PCR products were used to confirm the absence of non-specific amplification products. Relative expression levels were calculated by subtracting the threshold cycle (Ct) values for UBQ10 from those of the target gene (to give ∆Ct) and then calculating 2^−∆Ct, where C_t refers to the cycle number at which the fluorescence rises above the set threshold qPCR. UBQ10 was chosen as the housekeeping reference⁴⁰.

Phylogenetic and Statistical Analyses

DNA sequences were obtained from the 1001 Genomes project (www.1001genomes.org). All statistical analyses were conducted with DPS software (Stirling Technologies Inc., China). Means were compared by the t test or the Fisher’s least significant difference test at P = 0.05.

Results

Distribution and correlation of different 21 traits under normal and high Mg²⁺

Distribution of 388 Arabidopsis accessions collected from the wild and available in the stock center or soon-to-be-released collections is shown in Fig. 1A. In general, high Mg²⁺ supply (10,000 μM) had an adverse effect on the growth of most natural accessions. This high Mg²⁺ concentration induced a large degree of phenotypic variation and thus was selected for the quantitative analysis of the natural variation of Arabidopsis in response to high Mg²⁺ stress (Fig. 1B). A subset of 388 accessions (250 K SNPs) was characterized phenotypically for multiple traits, including root emergence (RG), shoot emergence (SG), primary root length (PR), lateral root number (LR), epicotyl length (EL), rosette width (WD), fresh weight (FW) of the plants and fourteen nutrient traits, including absorption per plant and concentration of K, Ca, Mg, S, Fe, Mn and Na under normal and high Mg²⁺ supplies (see Table 1 and ‘Phenotype analysis’ in Materials and Methods).

Figure 1.

Worldwide diversity of 388 A. thaliana natural accessions and cultivation used in this study according to their reported country origins and frequency distributions of non-normalized data of all traits. (A) The data were exported for visualization and geographical mapping in Tableau Desktop 8.1 (http://www.tableausoftware.com/). (B) Cultivation for 388 Arabidopsis inbred lines at Zhejiang University, Hangzhou, China. Each line was treated with low (1 µM), normal (1,000 µM), and high Mg²⁺ (10,000 µM). Each line had four biological replicates, and the median value was used in the analysis. (C) Frequency distributions of non-normalized data of all traits in the 388 population. Blue and Red line indicates normal and high Mg²⁺, respectively.

Table 1.

Summary for 21 Arabidopsis traits.

Traits	Full name of the trait	Unit	Trait description
RG	Days to root germination	day	The number of days from seeding until emergence, with more than half of the seedlings having a first radicle.
PR	Primary root length	cm	After 8 days of growth under the two treatment conditions, the plants were flattened directly on agar and imaged using a camera. The primary root lengths and lateral root number were determined across the median seedling using ImageJ.
LR	Lateral root number
SG	Days to seed germination	day	The number of days from seeding until emergence, with more than half of the seedlings having the first cotyledon.
EL	Epicotyl length	cm	After 8 days of growth under the two treatment conditions, the plants were flattened directly on agar and imaged using a camera. The epicotyl length and rosette width were determined across the median seedling using ImageJ.
RW	Rosette width	cm
FW	Fresh weight of plants	mg	All fully-expanded seedlings without lesions were collected from each of 4 plants for each accession and weighed to obtain the fresh weight. The results are the average value across all available replicates.
K-A	The amount of potassium absorbed per plant	mg K/plant	Elemental analysis was performed with an ICP-MS (Agilent 7500a, USA). All samples were normalized to calculated weights as previously described. The ‘Nutrient absorption(A)’ was expressed as total amount of nutrient per plant; ‘Nutrient concentration (C)’ was calculated by total amount of nutrient per plant /plant fresh weight (FW). The results are the average value across all available replicates.
K-C	Potassium concentration per fresh weight	mg K/g (FW)
Ca-A	The amount of calcium absorbed per plant	mg Ca/plant
Ca-C	Calcium concentration per fresh weight	mg Ca/g (FW)
Mg-A	The amount of magnesium absorbed per plant	mg Mg/plant
Mg-C	Magnesium concentration per fresh weight	mg Mg/g (FW)
S-A	The amount of sulfur absorbed per plant	mg S/plant
S-C	Sulfur concentration per fresh weight	mg S/g (FW)
Fe-A	The amount of iron absorbed per plant	mg Fe/plant
Fe-C	Iron concentration per fresh weight	mg Fe/g (FW)
Mn-A	The amount of manganese absorbed per plant	mg Mn/plant
Mn-C	Manganese concentration per fresh weight	mg Mn/g (FW)
Na-A	The amount of sodium absorbed per plant	mg Na/plant
Na-C	Sodium concentration per fresh weight	mg Na/g (FW)

Before performing the quantitative genetic analyses, we assessed the distributions of all traits based on all raw data (Supporting Information Table S1). The phenotypic analysis of the 388 accessions showed that the frequency distribution of all traits was close to normality (Fig. 1C; Table 1). Further detailed analysis showed that distribution of Mg-A-H (Mg²⁺ absorption under high Mg²⁺) and Mg-C-H (Mg²⁺ concentration under high Mg²⁺) showed a great right-skewed probability density with the right-skewed being greater for Mg-C-H than that for Mg-A-H, suggesting that high Mg²⁺ supply increased Mg concentration of most Arabidopsis accessions at least in the early phase of development. However, only slight skewness and kurtosis was observed for S absorption and concentration (Fig. 1C). Distribution of RW, EL, PR, FW, Ca-A (Ca absorption), Ca-C (Ca concentration), Na-A (Na absorption) and Na-C (Na concentration) presented a standard normal distribution but with a sharp peak upon high Mg²⁺ as compared with normal Mg²⁺ (Fig. 1C), indicating that the high Mg²⁺ treatment resulted in a more focused response in those traits. Notably, the right side of the curve of absorption and concentration of Ca, Fe and Mn under both normal and high Mg²⁺ fitted a normal distribution, but the left-hand side deviated significantly from the normal distribution, displaying a decapitation tail (Fig. 1C).

Data of the traits are close to normal distribution reflecting the homogeneity of the investigated population. However, what was the response of each individual domain to high Mg²⁺? According to the date (Supporting Information Table S1), the effects of high Mg²⁺ on all phenotypes can be divided into two types. First, high Mg²⁺ had a similar promoting effect on LR, Mg-A, Mg-C, S-A and S-C of most accessions. Second, high Mg²⁺ enhanced traits of PR, EL, RW, FW and absorption and concentrations of K, Ca, Fe and Mn in the accessions with low value while reduced them in the accessions with high value as compared with normal Mg²⁺ supply. Furthermore, the initial comparison of 21 plant traits between normal and high Mg²⁺ was performed using statistical analysis by a paired two-tailed Student t test, with a P-value < 0.01 (Table 2). Accordingly, the above first type traits were consistently changed by high Mg²⁺ as compared with normal Mg²⁺. Apart from this, the above second traits were inconsistently altered by high Mg²⁺ (Table 2, Fig. 2A,B).

Table 2.

Minimum (Min), maximum (Max), mean and standard deviation (SD) values of individual traits for 21 phenotypes under high (10,000 µM) and normal Mg²⁺ (1,000 µM).The correlations (r²) between values of individual traits of the plants grown at high and normal Mg²⁺ are also shown.

Normal Mg2+	Min	Max	Mean	SD	p-value (normality test)a
RG	1.5000	7.0000	2.4000	0.8200	1.07E-28
PR	0.1155	4.6749	1.6712	1.0430	4.61E-10
LR	0	9.0000	2.4845	1.7004	2.00E-16
SG	2.0000	7.0000	2.8814	0.7949	6.07E-22
EL	0.1305	1.1846	0.4088	0.1590	1.73E-12
RW	0.1493	1.2021	0.5823	0.2241	7.13E-05
FW	0.1000	4.025	1.2242	0.7383	3.26E-11
K-A	0.2105	14.185	3.6072	2.3203	8.73E-11
K-C	0.4935	8.5100	2.9159	0.9256	2.17E-11
Ca-A	0.0010	4.0225	0.5932	0.6245	2.49E-22
Ca-C	0.0009	7.4220	0.5646	0.8206	1.01E-31
Mg-A	0.0033	1.3830	0.3042	0.2049	6.52E-13
Mg-C	0.0118	1.5367	0.2547	0.1369	1.50E-27
S-A	0.0030	3.3967	1.0466	0.6320	1.62E-10
S-C	0.0300	6.7483	0.9736	0.6864	1.85E-28
Fe-A	0.0008	3.3113	0.1664	0.2977	1.45E-32
Fe-C	0.0007	8.7300	0.1826	0.5313	4.24E-37
Mn-A	0.0000	0.1647	0.0075	0.0134	2.73E-33
Mn-C	0.0000	0.3088	0.0084	0.0201	7.84E-36
Na-A	0.0300	5.6400	1.4760	1.2287	2.86E-21
Na-C	0.0643	21.200	1.4744	1.6282	3.50E-31
High Mg 2+	Min	Max	Mean	SD	p -value (normality test) a	r 2 (H vs N)	p -value (t-test) b
RG	1.5000	7.0000	2.4526	0.8379	2.54E-27	0.53	0.3607
PR	0.3301	3.5816	1.4777	0.6374	5.06E-10	0.3115	0.0019
LR	0.0000	7.0000	1.4845	1.3780	2.51E-17	0.4146	1.86E-18
SG	2.0000	7.0000	2.9021	0.7731	2.05E-20	0.5337	0.7143
EL	0.1327	0.8300	0.3825	0.1190	6.46E-10	0.2886	0.0092
RW	0.1111	1.3434	0.5714	0.1754	0.000113	0.2034	0.4492
FW	0.0500	3.4500	1.0945	0.5354	4.64E-12	0.3430	0.0052
K-A	0.0413	12.0925	3.4778	2.0909	2.89E-12	0.1070	0.4147
K-C	0.0148	17.2500	3.3030	1.4688	1.05E-20	0.0102	1.3130
Ca-A	0.0003	22.1050	0.3740	1.1442	1.54E-38	0.0057	0.0010
Ca-C	0.0002	36.8417	0.4576	1.9330	5.79E-39	0.0023	0.3163
Mg-A	0.0048	3.0103	0.9847	0.5822	2.78E-11	0.1679	2.13E-73
Mg-C	0.0021	3.7629	0.9260	0.3694	2.96E-19	0.0204	3.08E-129
S-A	0.0063	4.3700	1.3066	0.6900	6.40E-08	0.1911	6.01E-08
S-C	0.0015	4.3800	1.2760	0.5470	4.75E-12	0.0307	2.34E-11
Fe-A	0.0010	7.0500	0.2013	0.4963	2.72E-37	0.1023	0.2353
Fe-C	0.0007	4.2090	0.2164	0.3608	4.00E-33	0.0222	0.3012
Mn-A	0.0000	1.1415	0.0085	0.0585	1.17E-39	0.0224	0.7351
Mn-C	0.0001	0.6815	0.0077	0.0363	4.85E-39	0.0101	0.7021
Na-A	0.0025	4.7840	0.7881	0.5499	1.24E-21	0.0520	6.06E-22
Na-C	0.0011	12.2300	0.8342	0.7908	8.93E-31	0.0119	9.15E-12

^aSharipo test was used for normality test; ^bHigh and normal date-sample student’s t-test at p value < 0.01.

Figure 2.

Natural variation in traits of Arabidopsis accessions grown at normal (1,000 µM) (A) and high Mg²⁺ (10,000 µM). (B) The average value per accession was calculated, and values of individual traits were plotted using the ‘ggplot2’ package in R. Accessions representing outliers for a trait are indicated by closed circles. (C) Heat map represents cluster of the pearson correlation coefficient between each pair of 42 phenotypes, the pearson’s correlation coefficients were normalized (z-score) and represented with blue-red color scheme.

Pearson’s correlation coefficients (r²) were used to examine the natural variation within the population of quantified traits between normal and high Mg²⁺ conditions (Table 2). The RG and SG showed positive correlations between normal and high Mg²⁺ treatments (r² = 0.53) (Table 2). A weak correlation was observed for nutrient traits, especially Ca, Fe and Mg concentrations between normal and high Mg²⁺ treatments (r² < 0.2), suggesting different physiological functions of nutrients under different Mg²⁺ supplies.

Cluster of Pearson’s correlation coefficient between phenotypes (Heat map) showed a negative correlation between Ca-C-H, Fe-C-H and Na-C-H and Mg-C-H (Fig. 2C), suggesting that Mg²⁺ would compete for uptake with Ca²⁺, Fe²⁺ and Na²⁺ in most Arabidopsis accessions when supplied with high Mg²⁺. Increased Mg²⁺ absorption would decrease the uptake of these ions and hence plant growth. However, Mg-A, Mg-C, S-A, S-C, K-A and K-C were positively correlated only under high Mg²⁺ (Fig. 2C), indicating a common mechanism or genetic pathway between Mg²⁺, S and K under high Mg²⁺ (Fig. 2C). In contrast, there were highly significant positive correlations between PR, RW, LR, FW as well as S-A, Mg-A and K-A of the phenotypes under normal but not high Mg²⁺ supply (Fig. 2C).

GWAS analysis for nutrient metabolism in response to high Mg²⁺

The phenotypic data collected from normal and high-Mg²⁺ conditions (Supporting Information Table S1) were used as an input for genome-wide association study (GWAS). All SNPs were converted to homozygote or heterozygote according to the genotype, and tri-allelic or tetra-allelic SNPs were removed. Candidate loci were selected based on the minor allele frequency, LOD score and number of significantly associated SNPs within one locus. Using −log₁₀(p) > 6.15 as the nominal genome-wide significance threshold, the GWASs had 109 hits in total, 32, 10, 25, 18 and 24 located on chromosomes (Chr) 1, 2, 3, 4, and 5, respectively, under normal Mg²⁺, and 79 hits in total, 14, 13, 18, 10 and 19 located on chromosomes 1, 2, 3, 4, and 5, respectively, under high Mg²⁺ (Fig. 3A,B). There were 2 shared hits between normal and high Mg²⁺ supplies (Fig. 3C).

Figure 3.

GWASs for 21 traits under normal (1,000 µM) and high Mg²⁺ (10,000 µM) conditions. The x-axis is the chromosomal coordinates for Arabidopsis, the y-axis represents p-value. The GWASs hits observed at high Mg²⁺ supply were presented on the top; the hits observed at normal Mg²⁺ were represented at the bottom panel (A). GWAS hits and chromosome were represented by different symbols and coloured, respectively. GWAS hits were shared between normal and high Mg²⁺ were ordered by their longitudinal (x-axis) position. A SNP GWAS hit was defined as the one had −log10(p) > 6.15. (B) Total number of traits that were scared for SNPs with log10(p) > 6.15. The x-axis represents scared traits for Arabidopsis, the y-axis is the p-value. Blue and Red lines represent normal and high Mg²⁺, respectively. (C) Overlap between total number SNP hits for 21 traits at normal and high Mg²⁺. Venn diagrams display overlap between SNP number at normal Mg²⁺ (blue) and high Mg²⁺ (red).

Under normal Mg²⁺ supply, 41 and 22 loci were associated with Mn absorption (Mn-A-N) and concentration (Mn-C-N), and accounted for 7.47% and 2.67% of the total phenotypic variance, respectively. 17 and 21 loci were associated with Fe absorption (Fe-A-N) and concentration (Fe-C-N), and accounted for 3.60% and 5.04% of total phenotypic variance individually (Fig. 3B, Supporting Information Table S3). In comparison, under high Mg²⁺ supply, 16 and 13 loci were associated with Mn absorption (Mn-A-H) and concentration (Mn-C-H), and accounted for 2.93% and 12.04% of total phenotypic variance individually. Thirty three and 11 loci were associated with Fe absorption (Fe-A-H) and Fe concentration (Fe-C-H), which accounted for only 5.07% and 2.60% of total phenotypic variance individually (Fig. 3B, Supporting Information Table S2). The GWAS analysis confirmed that only 2 common loci were detected differed between normal Mg²⁺ and high Mg²⁺ (Fig. 3C).

Single nucleotide polymorphisms (SNP) 1_3884526 (Chromosome 1, 3884526 bp) was found to associate with SG-H, RG-H, RG-N and Na-C-N. It had a Minor allele frequency (MAF) of 0.0633, and is within the OST3A, a gene related to oligosaccharyl transferase complex/magnesium transporter family (Supporting Information Table S2). SNP 4_2080234 was associated with Mn-C-H and Fe-C-N. It had a MAF of 0.050 and is within AT4G042960 and AT4G04293, both transposable element genes.

No significant associations were observed for the Mg²⁺ (Mg-A) and Ca absorption (Ca-A) under normal Mg²⁺, but 2 loci were identified to associate with these traits under high Mg²⁺. Furthermore, no significant associations were found for Mg²⁺ (Mg-C) and S concentration (S-C) under high Mg²⁺, whereas 7 and 6 loci were identified to associate with these traits under normal Mg²⁺ (Fig. 3B). No significant associations for K absorption (K-A) were identified under normal and high Mg²⁺.

We speculated that the absorption and concentration of nutrients should share many candidate loci associated with the same element under the same Mg²⁺ level. However, this was not the case in our data set, as no common candidate loci were observed to associate with absorption and concentrations of K, Ca, Mg, S and Na under either normal or high Mg²⁺ (Supporting Information Table S2). Though common loci were found between the absorption and concentration of Fe or Mn in plants grown under both normal and high Mg²⁺, these only accounted for a small part of the variance in attitudes. While six common loci were detected between the traits of Fe-A-H and Fe-C-H, only 2 common SNPs were for Fe-A-N and Fe-C-N. Likewise, 10 common loci were associated with Mn-A-H and Mn-C-H and 6 common loci associated with Mn-A-N and Mn-C-N (Fig. 4A,B). Furthermore, manhattan plot showed the detailed distribution of the GWAS hits for Fe and Mn (Fig. 4C,D).

Figure 4.

Venn diagrams representing differentially nutrient (including absorption and concentration) GWAS hits was defined as the one had −log10(p) > 6.15 in high Mg²⁺ (A) and normal Mg²⁺ (B) of Arabidopsis. Each list in the venn diagram denotes by a transparent shape and overlaps shape indicates elements shared between lists or more often the corresponding counts. Four lists of input data of Fe-A, Fe-C, Mn-A and Mn-C were highlighted for both high Mg²⁺ and normal Mg²⁺ in green, blue, pink and yellow, respectively. Genome-wide association mapping results for the absorption and concentration of Fe and Mn in Arabidopsis under high Mg²⁺ (C) and normal Mg²⁺. (D) The ‘Manhattan setting’ plate for customized chromosome color, fixed pixel size, or dynamic pixel size proportional to p-values in Manhattan plot. The 5 chromosomes are represented by different shades of gray with the x-axis as the physical position. The highlighted positions represent the physical positions of the regions sharing common SNP between absorption and concentration of Fe and Mn.

Characterization of Atntrl mutants in response to high Mg²⁺ treatment

GWAS is conducted to identify causative/predictive factors for a given trait, or to determine aspects of the genetic architecture of the trait (i.e. the number of loci that contribute to the phenotype)⁴¹. A single strong peak of SNPs 22262848 between position of 22262147 and 22266184 bp on chromosome, which is associated with Ca concentration, was identified under high but not normal Mg²⁺ (Fig. 5A,B). This SNP accounted for 0.326% of the total variance of an estimated 2.20% of the total variance in Ca concentration under high Mg²⁺. It contained three neighboring significant SNP hits (−log₁₀(p) > 6.9) corresponding to only a candidate gene At1g60420 (AtNRX1) (Fig. 5C), and was calcium locus CD-domain containing protein.

Figure 5.

Manhattan plot illustrating the GWAS mapping of the Ca concentration in 388 Arabidopsis accessions grown under supply of normal (1,000 µM) (A) and high Mg²⁺ (10,000 µM). (B) Chromosomes are shown in different colors. (C) Detailed plot of the region shown in the red box in (B). The positions on the chromosome are on the x axis and the score on the y axis. The dots in the scatterplot represent SNPs. A horizontal dashed line shows the 5% threshold.

To validate the GWAS results in the present study, the above putative significant candidate gene was determined through a collection of well-characterized mutants (Atnrx1-1 and Atnrx1-2) and physiological and morphological comparisons in mutants and wild-types. Before this, we further verified that the mutant stocks were homozygous T-DNA insertion lines, by PCR with gene-specific primers LP, RP and LB. As expected, the results confirmed that both Atnrx1-1 and Atnrx1-2 were only mutation in At1g60420 (Fig. 6A). Moreover, the expression of AT1G60420 were greatly reduced in two mutants under both normal and high Mg²⁺ conditions as compared to wildtype (Fig. 6B). Compared with normal Mg²⁺, high Mg²⁺ increased the fresh weight of Atnrx1-1 and Atnrx1-2 but did not affect that of wildtype. It is worth mentioning that compared with wildtype, fresh weight of two mutants was not changed under high Mg²⁺ but was reduced under normal Mg²⁺ (Fig. 6C). It is suggested that high Mg²⁺ reduced wildtype growth but had little effect on the growth of Atnrx1 mutant.

Figure 6.

Validation and physiological phenotypes of Atnrx1 mutant. Image of mutant Atnrx1-1 and Atnrx1-2 and wild plants in media with normal or high Mg²⁺ (A); results of T-DNA insertion mutant Atnrx1-1 and Atnrx1-2 by PCR with gene-specific primers LP, RP and LB (B); relative expression levels (C); fresh weight (D); confocal images of cytosolic Ca²⁺ concentration distribution (Fluo-4/AM imaging) during the initiation and tip growth of root hairs (E); concentrations of Ca (F); concentrations of Mg²⁺ (G); number of lateral root per plant (H) and the length of primary root (I) of wild-type and Atnrx1 mutant (8-day-old seedlings) grown at supply of normal (1,000 µM) and high Mg²⁺ (10,000 µM). Levels of cytosolic Ca²⁺ concentration have been color-coded according to the inset scale (Quantitative values under the scale in nM). Bar = 200 μm applies to all photographs in D. The images are representative of at least three independent experiments. Data are means ± SD (n = 5). Means followed by a common letter within a root segment are not significantly different at P < 0.05. FW, fresh weight.

As shown in Fig. 6D, a tip-focused [Ca²⁺]_c gradient was formed during the initiation and elongation of roots of both wildtype and mutants, and such a tip-focused gradient of [Ca²⁺]_c was weak under normal Mg²⁺. In contrast, under high Mg²⁺, Atnrx1-1 and Atnrx1-2 but not wildtype formed a strong tip-focused [Ca²⁺]c gradient in the initiation and elongation of root of the mutant (Fig. 6D). Furthermore, high Mg²⁺ greatly enhanced concentration of Ca in Atnrx1-1 and Atnrx1-2 but decreased it in wildtype as compared with normal Mg²⁺ (Fig. 6E). Meanwhile, when compared with wildtype, Ca concentration of high Mg-fed Atnrx1-1 and Atnrx1-2 mutants was greater while that of normal Mg-fed Atnrx1-1 and Atnrx1-2 mutants was similar (Fig. 6E). As expected, high Mg²⁺ enhanced Mg concentrations in both mutants and wildtype (Fig. 6F).

Compared with the wildtype, both Atnrx1-1 and Atnrx1-2 had shorter primary root and less number of lateral roots under normal Mg²⁺ but had greater length of primary roots and a similar number of lateral roots under high Mg²⁺ (Fig. 6G,H). Under high Mg²⁺, there is no significant difference in K, Fe, Mn and S concentrations in plants between the two mutants and wildtype, but the mutant had a higher Na content (Data not shown). These results indicate that AtNRX1 is a special and critical negative gene regulating Ca uptake and probably the plant growth under high Mg²⁺ stress.

Discussion

To adapt to the constantly high availability of Mg²⁺ in the environment, plants have evolved mechanisms of reduced ion uptake and/or sequestering excessive ions in the vacuole or developing higher Mg²⁺ requirement for the maximum growth, which finally improves the tolerance to the stress of certain elements. By linking the mapping with reverse genetic experiments, we identified the causal gene and established that polymorphisms at AtNRX1 were the major genetic determinant for the variation in Ca concentration in this global A. thaliana population (Fig. 6). Atnrx1 mutants have most of the phenotypes that would associate with the tolerance to serpentine soils, including survival in solutions with a low Ca²⁺: Mg²⁺ ratio; requirement for a high concentration of Mg²⁺ for maximum growth; greater biomass and more lateral roots, reduced leaf tissue concentration of Mg; and poor growth performance on ‘normal’ levels of Mg²⁺.

Shoot and root morphology variation in response to high Mg²⁺

In order to understand molecular mechanisms of plant adaptation to high Mg²⁺ stress, we grew 388 wild Arabidopsis accessions at normal and high Mg²⁺ concentrations, and observed a substantial quantitative variation in plant growth between these accessions (Fig. 1; Table 1). We also showed a diversity in shoot and root morphology across two levels of Mg²⁺ supply and among these natural accessions. High Mg²⁺ supply resulted in an overall reduction in primary root length, lateral root number, epicotyl length and fresh weight but its effect on the rosette width varied among the accessions (Fig. 2; Table 2). The phenotypic data indicate that Arabidopsis thaliana has evolved some capacities to adapt to high Mg²⁺ stress and that the number of lateral roots of seedlings is a suitable index of high-Mg²⁺ responses.

By using the phenotypic data of 388 Arabidopsis accessions (Supporting Information Table S1) and GWAS, this study showed that a great extent of variation in the length of primary and lateral roots, rosette growth and hypocotyl length among the accessions tested was strongly Mg-dependent. As we known, the growth of primary and lateral roots, hypocotyl and rosette is determined by a number of extrinsic and intrinsic factors in addition to Mg²⁺ supply. The significant associations with the leaf or root growth-related phenotypes according to −log₁₀(p) > 6.15 reflects the high complexity of the underlying genetic architecture or that these traits were not influenced by any of the SNPs alone. This notion is supported by the presence of obvious sub-threshold peaks associated with shoot and root morphology under both normal and high Mg²⁺ supply. Meanwhile, the low heritability of investigated traits in the present study suggested a weak genetic component determining the observed phenotypic variability. Hopefully, it is known that many traits of Arabidopsis are polygenic with small effect size, and that increasing the sample size would improve the power to recover meaningful associations⁴². Therefore, increasing sampling size was likely to underestimate the range natural variation in photomorphogenic responses or growth-related pathways and processes that strongly interact with the high Mg²⁺ stress.

Arabidopsis showed remarkable natural genotypic variation for ionomics under high Mg²⁺

The present study showed that most of the SNPs were derived from the nutrient concentration and uptake, SNPs differed between normal and high Mg²⁺, the effective number of SNPs (−log₁₀(p) > 6.15) was also different (Fig. 3; Supporting Information Table S2). For example, positive SNPs associated with Ca-A and Mg-A were located only under high Mg²⁺ (Fig. 3B). Likewise, no significant associations were observed for either Mg-C and S-C under high Mg²⁺ but seven and six significant SNPs were associated with them under normal Mg²⁺ (Fig. 3), indicating that different genetic systems were responsible for Mg²⁺ and S uptake under normal and high Mg²⁺. Meanwhile, the SNP significance tests showed that different traits were generally associated with different genes or gene regions, but many of the traits also shared common genes or gene regions. The significant score >10 association was for the SNP on Mn under high Mg²⁺ in a narrow region of chr3, where multiple SNPs were associated with different traits in Chr1, 3, 4 and 5 under normal Mg²⁺ (Fig. 3).

Though not all cases, the chromosomal regions harbouring the significantly associated SNPs are coincided with the location of the gene(s) co-expressed with known-function genes that control these traits. Of the 105 loci under normal Mg²⁺, the majority of the loci were associated with Mn-A and Mn-C then with Fe-A and Fe-C. Among these, SNP 1_3953597, which is within the SS3, a gene encoding a starch synthase, is co-expressed with SPLICING ENDONUCLEASE 1 (SEN1)⁴³. Likewise, SNP 3_19424421 of p-value 12.55, associated with Mn-C under the normal Mg²⁺ condition, had a MAF of 0.058 and was within gene AT3G52380 CHLOROPLAST RNA-BINDING PROTEIN 33 (CP33) that is co-expressed with SEN1. SEN1 gene was reported encoding an integral membrane protein homologous to Glycine max nodulin-21, and also to CCC1, a vacuolar Fe/Mn transporter of Saccharomyces cerevisiae of Arabidopsis⁴¹. Besides, SNP 1_210424, associated with Fe-C, had a MAF of 0.055 and was within gene FRO2 which encodes the low-Fe-inducible ferric chelate reductase. It is suggested to be the major Fe(III) chelate reductase in Arabidopsis⁴⁴. The most SNP hits under high Mg²⁺ were identified associations with Fe-A and Fe-C and then Mn-A and Mn-N. SNP 1_26930918 had a MAF of 0.074 and was located within AT1G71480 and AT2G33410 genes which are involved in nuclear transport factor 2 family gene, and are co-expressed with Fe transporter gene FERRIC REDUCTION OXIDASE 6 (FRO6) and 7 (FRO7)⁴⁵ and 1 (FRO1)⁴⁶. Furthermore, SNP 2890675, associated with Fe-C under the high Mg²⁺ on Chromosome 5, had a MAF of 0.074 and was located within AT5G09310 gene and co-expressed with Fe transporter gene FRO1⁴⁶. Likewise, SNP 3_6977829 of P-value 8.40, associated with Fe-A, within gene AT3G20015 (ASPG2), and was co-expressed with H( + )-ATPASE 2 (AHA2), a main actor in Fe uptake and signaling⁴⁷.

In addition, SNP 4_772594 associated with Mg + C under the normal Mg²⁺, had a MAF of 0.079 and was located within AT4G01800 ALBINO OR GLASSY YELLOW 1 (AGY1) gene which was co-expressed with MAGNESIUM CHELATASE I2 (CHLI2) while CHLI2 regulates the function of Mg²⁺ chelatase⁴⁸. It should be mentioned that SNP 1_21762851 of P-value 9.05, associated with S-C under the normal Mg²⁺, had a MAF of 0.090 and contained only one gene, AT1G51980, which encodes an insulinase and is co-expressed with SULFATE TRANSPORTER 1;2 (SULTR1;2) that is involved in sulfur metabolism⁴⁹. As discussed above, changing the Mg²⁺ availability in the soil would alter absorption and utilization of other elements, and transporters, channels, or/and the genes that encode and regulate them in Arabidopsis. However, a few of the genes in proximity of the most significant associations were among the major known regulators of ion uptake or transport, highlighting the potential of GWAS to identify previously unknown regulators⁵⁰.

Changes in nutrient availability influence plant vegetative growth and phenotypic traits of A. thaliana, but the effect may vary among populations or genotypes^51–53. Decreases in nutrient availability result in phenotypic responses (such as smaller leaf areas, shorter primary root and reduced lateral root formation) that vary across A. thaliana genotypes.

A novel candidate gene AtNRX1 negatively regulates Ca concentration at high Mg²⁺ supply

Calcium is an essential plant macronutrient with key structural and signaling roles. Calcium ions (Ca²⁺) act as an osmoticum within vacuoles, a stabilizing element in the membranes, a strengthening agent in cell walls, and a secondary messenger for a multitude of signals^54–56. By using GWA mapping on the 388 A. thaliana accessions, we successfully identified a single strong peak of SNP 1_22263976/22264331/22262848 (Chromosome 1, 22263976 bp) associated with Ca concentration under high Mg²⁺ (Fig. 5). Mutants of Atnrx1-1 and Atnrx1-2 supplied with high Mg²⁺ had higher cytosolic Ca²⁺ concentrations ([Ca²⁺]_c) during root elongation, meanwhile produced higher biomass and more lateral roots as compared with wildtype (Fig. 6). This suggested that the increase in Ca concentration in mutants of Atnrx1-1 and Atnrx1-2 by high Mg²⁺ cannot be attributed to increased biomass or photosynthesis alone, but also to the improved Ca nutrition of the plants through morphological, physiological and molecular responses to high Mg²⁺.

It has been known that AtNRX1 is involved in DC1 domain-containing protein, which is co-expressed with calcineurin B-like Ca sensor 1–protein kinase gene CBL-INTERACTING PROTEIN KINASE 23 (CIPK23)^57,58. Study of Tang et al.⁵⁹ showed that tonoplast CBL–CIPK calcium signaling network regulated ion homeostasis and vacuolar sequestration of Mg²⁺, thereby protecting plants from Mg²⁺ toxicity^59,60. Besides, loss-of-function cax1 (gene encoding vacuolar H⁺/Ca²⁺) mutations could produce phenotypes characteristic of plants adapted to serpentine⁸.

This locus has a MAF of 0.0712 and is within the AT1G60420 (AtNRX1), a gene involved in catalytic activity of NADP-NADPH and disulfide-reductase activity⁶¹. In special cases, toxic ROS molecules are largely accumulated under excess stresses and diverse enzymes played as ROS scavengers. Arabidopsis contains 3 NADPH-dependent thioredoxin reductases (NTRs) for scavenging ROS and Nucleoredoxins (NRX) are potential nuclear TRX found in most eukaryotic organisms⁶¹. Recently, excellent research presents evidence that activity of the plant stresses-inducible TRX superfamily member, NRX1 is necessary for the integrity of antioxidant systems⁶². Meanwhile, it is the fact that many publications indicated ROS produced by NADPH oxidase activates Ca²⁺ channels at the plasma membrane, leading to an increase in Ca²⁺ concentration and a tip-focused Ca²⁺ concentration gradient and subsequent signaling inherent to plant growth^32,63–66. Withal, our previous study also showed that high Mg²⁺ could inactivate NADPH and the production of ROS²⁷ probably through regulating ABA–DELLA signaling^28,67.

To sum up, it is likely that under high Mg, the wild-type plant is associated with cellular accumulation of ROS and redox imbalance, which triggers class of antioxidant enzymes activation and subsequently results in a decrease of intracellular ROS. ROS restrains ion channels and Ca²⁺ concentration; While in the nrx1 mutants, ROS could not be effectively removed without protection of NRX1 due to NRX1 mutation, which leads to ROS accumulation. Then ROS activate Ca²⁺-channels at the plasma membrane and promote cytosolic Ca²⁺ concentration. When both wild-type and nrx1 mutants under normal Mg conditions, the cell is normally controlled with H₂O₂ and redox balance, thus the protective effect of NRX1 gene is not prominent, or maybe even little dedication.

Notably, Mg²⁺ concentrations in Atnrx1-1 and Atnrx1-2 mutants was increased compared to that in the wildtype plants under high Mg²⁺ supply. This implied that nrx1 mutants required for a high concentration of Mg²⁺for the maximum growth associating with tolerance to high Mg²⁺ soils. The AtNRX1 gene is co-expressed with MAGNESIUM TRANSPORTER 4 (MRS2-3), a transport gene related to Mg²⁺ nutrition⁶⁸. These indicate that Mg²⁺ transporter genes have some interactions with AtNRX1. However, there is no significant difference in K, Fe, Mn and S concentration in plants between the two mutants and wildtype, but the mutant had a higher Na content (Data not shown). These was consistent with other study showed that the cbl2 cbl3 double mutant was hypersensitive to a handful cations but not to Na^+ 59. Further work will be directed to identify other key components in this novel of NRX-signaling pathway in regulation of Ca uptake and how the positive effects of AtNRX1 can be maximized through plant adaptive strategies to improve the tolerance to high Mg²⁺.

To sum up, our study suggests that AtNRX1 was a critical gene negatively regulating Ca uptake and probably improving the plant tolerance to high Mg²⁺ soils. The discovery of the functions of AtNRX1 gene helps to breed/select crops that can adapt to high-Mg²⁺ soils such as serpentine soils (high Mg²⁺: Ca) and those in semi-arid regions or even serpentine or Mars soils with high levels of magnesium sulfate. Also, new genes associated with natural variation in A. thaliana can be used for future bioengineering.

Author Contributions

Y.F.N. developed the concept and designed the study; Y.F.N., P.C. and Y.Z. performed the experiments; Y.F.N. and G.L.J. analyzed the data; Z.W.W. and S.K.H. interpreted the results of the experiments; C.X.T. and L.B.G. edited and revised the manuscript; All authors approved the final version of the manuscript.

Competing Interests

The authors declare no competing interests.

Electronic supplementary material

Supplementary information accompanies this paper at 10.1038/s41598-018-31950-0.