1365313x, 2023, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/tpj.16042 by Readcube (Labtiva Inc.), Wiley Online Library on [01/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License The Plant Journal (2023) 113, 225–245 doi: 10.1111/tpj.16042 A genome-wide association study identifies novel players in Na and Fe homeostasis in Arabidopsis thaliana under alkaline-salinity stress Maria Jose Almira Casellas1,† , Laura Perez-Martın1,2,† , Silvia Busoms1 , Rene Boesten3 , Merce Llugany1 , 3,* 1,* Mark G. M. Aarts and Charlotte Poschenrieder 1 Plant Physiology Laboratory, Bioscience Faculty, Universitat Auto noma de Barcelona, C/de la Vall Moronta s/n, E-08193, Bel- laterra, Spain, 2 Department of Botany and Plant Biology, University of Geneva, 1211, Geneva, Switzerland, and 3 Laboratory of Genetics, Wageningen University and Research, Droevendaalsesteeg 1, 6708 PB, Wageningen, The Nether- lands Received 26 October 2022; revised 11 November 2022; accepted 21 November 2022; published online 17 December 2022. *For correspondence (e-mail charlotte.poschenrieder@uab.es and mark.aarts@wur.nl) †† These authors contributed equally. SUMMARY In nature, multiple stress factors occur simultaneously. The screening of natural diversity panels and subse- quent Genome-Wide Association Studies (GWAS) is a powerful approach to identify genetic components of various stress responses. Here, the nutritional status variation of a set of 270 natural accessions of Ara- bidopsis thaliana grown on a natural saline-carbonated soil is evaluated. We report significant natural varia- tion on leaf Na (LNa) and Fe (LFe) concentrations in the studied accessions. Allelic variation in the NINJA and YUC8 genes is associated with LNa diversity, and variation in the ALA3 is associated with LFe diversity. The allelic variation detected in these three genes leads to changes in their mRNA expression and correlates with plant differential growth performance when plants are exposed to alkaline salinity treatment under hydroponic conditions. We propose that YUC8 and NINJA expression patters regulate auxin and jasmonic signaling pathways affecting plant tolerance to alkaline salinity. Finally, we describe an impairment in growth and leaf Fe acquisition associated with differences in root expression of ALA3, encoding a phospho- lipid translocase active in plasma membrane and the trans Golgi network which directly interacts with pro- teins essential for the trafficking of PIN auxin transporters, reinforcing the role of phytohormonal processes in regulating ion homeostasis under alkaline salinity. Keywords: alkaline-salinity, GWAS, Arabidopsis, natural variation, ionome. INTRODUCTION pattern and environment specificity of adaptive variants Plant responses to abiotic stress are presumably shaped and the type of genes involved. by a combination of adaptation to the local environment, The diversity of stress responses present within one accumulation of mutations, gene flow between the popula- species can be harnessed by screening natural diversity tions and random effects. Local adaptation is particularly panels and performing subsequent Genome-Wide Associa- relevant in the face of the global changes our planet is tion Studies (GWAS) to identify genetic components of exposed to, because locally adapted populations can various stress responses, as has been done in the past for become maladapted if environmental conditions change e.g., salinity (Baxter et al., 2010; Katori et al., 2010; Rus rapidly (Davila Olivas et al., 2017). When studying the et al., 2006), carbonates (Perez-Martın, 2020; Teres, 2017) genetic mechanisms and the extent of local adaptation, or combinations of stress factors (Davila Olivas et al., 2017; understanding the genetic basis of fitness variation across Kawa et al., 2016). GWA studies enumerate underlying different ecological conditions is of major importance. This SNPs (single nucleotide polymorphisms) responsible for includes identifying the loci associated with individual fit- certain phenotypic traits by exploiting the natural variation ness in different natural environments, the distribution in a population or collection of populations of the studied Ó 2022 The Authors. 225 The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. 1365313x, 2023, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/tpj.16042 by Readcube (Labtiva Inc.), Wiley Online Library on [01/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 226 Maria Jose Almira Casellas et al. species (Ogura & Busch, 2015; Weigel, 2012). More specifi- et al., 2018; Mwando et al., 2020), soybean (Do et al., 2019; cally, GWAS evaluate the statistical significance of associa- Jin et al., 2021; Zeng et al., 2017; Zhang et al., 2019), cotton tions between differences in a quantitative phenotype and (Yasir et al., 2019), or wheat (Chaurasia et al., 2021) have genetic polymorphisms tested across many genetically dif- been cutting-edge studies performed in recent years. ferent individuals by using linkage disequilibrium (LD), Alkaline–sodic environments are defined by a high based on non-random association between alleles and OH concentration in the waters and soils accompanied by phenotypes (Uffelmann et al., 2021). GWAS identifies asso- the dominance of sodium base cations in both dissolved ciations between marker SNPs and phenotypes without and exchangeable forms. In this context, excess of dis- the need of constructing special mapping populations, and solved carbonate species is required for the development it uses high recombination in natural populations instead of alkalinity and the increase of the dispersive power of (Bazakos et al., 2017; He et al., 2017; Korte & Farlow, 2013). sodium (Jobbagy et al., 2017). Soil carbonates have been The candidate genes identified by GWAS can eventually much less explored through GWAS as a potential driving serve to guide marker-assisted breeding and the genetic agent of natural variation. Most studies focus on the defi- modification of crops (Li, 2020). In this regard, recent ciency of iron (Mamidi et al., 2014, Satbhai et al., 2017, reviews (Liu et al., 2020) point to GWA studies as an Assefa et al., 2020 and Xu et al., 2022, in Arabidopsis, increasingly successful analysis in the identification of key chickpea, soybean and maize, respectively) or phosphorus genes that are useful for agronomic traits of interest. (Liu et al., 2015 and Reddy et al., 2020, in mungbean) when In the model plant Arabidopsis thaliana, GWAS are a studying the effects of alkalinity stress. The cited studies powerful approach to identify loci associated with fitness lead to the identification of new candidate iron responsive and abiotic stress responses. Multiple growth and repro- genes such as AT1G01580/FRO2 (Mamidi et al., 2014; Satb- ductive life history traits showing natural variation along hai et al., 2017) and At5g53970/NAAT1, Os03g0237100/ geographical gradients such as flowering time, seed dor- DMAS1, GRMZM5G812538/YSL11, AT4G19690/IRT1, mancy, growth rate, resistance to abiotic stresses, defense AT1G10970/ZIP4 and AT2G25490/EBF1 (Xu et al., 2022). related traits, ionome profile or root growth have proven In nature, multiple stress factors occur simultaneously. to be good input data sets for GWA analyses. Atwell Thus, assessing how plants respond to a combination of et al. (2010) demonstrated for the first time the power of stresses is ecologically more representative of how their GWAS for describing the genetics of natural variation in performance will be under field conditions. In areas with such traits in A. thaliana, many of them of great agricul- arid and semi-arid climates, like the Mediterranean region, tural importance. soil salinity often co-occurs with alkalinity due to the move- Stress resistance is one of the most ecologically rele- ment of soluble ions like Na+, Ca2+, Mg2+, K+, CO32 and vant traits in a plant species. Due to its agronomic impor- HCO3 to the superficial soil layers produced by the tance, salinity is among the most studied abiotic stress increase in evapotranspiration rates (Singh, 2021). Alkaline- factors (Julkowska et al., 2014; Knight et al., 1997; Shabala salinity stress has gained importance, in the last decade, as et al., 2005). Since the start of GWAS in A. thaliana, impor- a major limiting factor for world crop production (Bai tant achievements have been obtained in revealing new et al., 2018; Huang, 2018; Zhao et al., 2021). Around 831 mil- genes and mapping new Quantitative Trait Loci (QTLs) for lion hectares of the Earth are covered by alkaline-saline soils salinity tolerance. For example, by means of association (FAO, 1973). Alkaline-saline stress exerts complex harmful mapping of ionome profile data of 394 Arabidopsis acces- effects on plants in the form of osmotic pressure, ion toxic- sions, Baxter et al. (2010) identified HKT1 as one of the ity, nutrient deficiency, and high pH (Liu et al., 2021). Alka- major contributing factors to natural variation in Na+ accu- line salts inhibit germination, growth, photosynthesis, and mulation in shoots. HKT1 was previously known to be root system activity, and are reported to increase shoot Na+ involved in sodium-driven potassium uptake (Rubio and ion imbalance with higher intensity than neutral salts in et al., 1995), sodium uniport in roots (Haro et al., 2005; Lau- Lathyrus quinquenervius (Zhang & Mu, 2009). When com- rie et al., 2002), differential Na+ accumulation in aerial parts pared to NaCl stress, alkaline-saline stress can increase the (Rus et al., 2006), and shoot Na+ exclusion and salt toler- activities of H+-ATPase and H+-VPPases, which are proton- ance (Møller et al., 2009). HKT1, together with CYTO- driven forces for Na transport at the cell and vacuolar mem- CHROME P450 FAMILY 79 SUBFAMILY B2 and B3 branes (Deinlein et al., 2014; Ye et al., 2019). Sodium (CYP79B2/B3) were later confirmed as new components sequestration into vacuoles takes place through SOS2/ contributing to lateral root hair development under salt SOS3 kinase-regulated SOS1 and NHX1, a process sensitive stress by performing GWAS on root traits from the Ara- to Ca2+ ions (Bahmani et al., 2015; Fang et al., 2021). Thus, bidopsis HapMap collection (Julkowska et al., 2017). GWA adequate Ca levels are essential under alkaline salinity. mapping of salinity tolerance in cultivars of interest like To a limited extent, alkaline salt tolerance has been rice (Kumar et al., 2015; Nayyeripasand et al., 2021; War- explored by means of association mapping in the last dec- raich et al., 2020), barley (Fan et al., 2016; Hazzouri ade. A major QTL allele from wild soybean was identified Ó 2022 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd., The Plant Journal, (2023), 113, 225–245 1365313x, 2023, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/tpj.16042 by Readcube (Labtiva Inc.), Wiley Online Library on [01/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License GWAS on alkaline-salinity responses in thaliana 227 for increasing alkaline salt tolerance (Tuyen et al., 2010). the accessions used in this study and the physicochemical Epistatic association mapping in 257 soybean cultivars properties of the SCS are described in Figure 1a,b. Selecting with 135 SSR markers was performed to assess salt toler- an adequate soil to mimic the conditions of representative ance under control, NaCl or Na2CO3 conditions and small- saline carbonated soils in Mediterranean areas was priori- effect QTLs were identified for further confirmation (Zhang tized. As soil-forming factors and dynamics are interdepen- et al., 2014). More recent association studies have provided dent processes that create a unique and high complex novel candidate locus to crop breeders for improving system, no commercial soil was considered reliable as a saline-alkaline adaptation (Cao et al., 2020). control and plant performance was only assessed on SCS. Here, we use a natural alkaline saline soil to assess the High natural variability was observed for rosette diam- variation in growth and nutritional status in a set of 270 eter (RD) of 8-weeks-old plants among all the accessions natural accessions of the widely distributed model species (Table S1). We classified the studied accessions according A. thaliana and we explore the molecular genetic mecha- to their RD into low diameter (LD) and high diameter (HD) nisms underlying possible stress responses to alkaline groups, based on the 10% tails of the RD normal distribu- salinity. In this study, soil complexity was embraced by per- tion. Differences in RD were statistically significant among forming GWAS on natural saline calcareous soil from a the two groups (Figure 2a). coastal Mediterranean region. There is high variation in the Elemental profiling of leaf tissue was performed for mineral nutrient accumulation among different populations three plants of each of the 270 studied accessions. High within a species depending on the chemical composition, variability was observed depending on the element and nutrient, and trace element bioavailability from their native the accession (Table S1). To understand the relationship soils (Baxter & Dilkes, 2012). Therefore, the natural varia- between the elemental concentrations, we performed mul- tion in the ionomic profiles of the studied A. thaliana acces- tivariate correlation analysis on the 10 quantified leaf min- sions was used as the main phenotype for this GWAS. eral nutrients and the rosette diameter traits and clustered the elements based on their degree of correlation. The RESULTS highest positive and negative correlations were found Figure S1 details the workflow and the A. thaliana acces- between leaf Fe - Mn (R2 = 0.78) and Fe - K (R2 = 0.64), sions and mutants used in each experiment. respectively. Leaf Fe concentrations were strongly corre- lated to several elements under SCS alkaline salinity condi- Arabidopsis thaliana HapMap performance on saline tions besides Mn and K and showed a positive correlation calcareous soil to Ca (R2 = 0.29) and negative correlation to S and P A total of 270 accessions constituting the HapMap collection (R2 = 0.27). Although to a less degree, leaf Na displayed (Horton et al., 2012) were grown for 8 weeks on a saline car- significant positive and negative correlations only with Fe bonated soil (SCS) excavated from a coastal region of the (R2 = 0.10) and S (R2 = 0.10). Na and S were the only mea- northeast of Spain (Figure 1a). The geographical location of sured elements showing a significant negative correlation Figure 1. Plant material and soil of study. (a) Distribution of saline and calcareous soils in Europe. Green dots indicate the location of origin of the 360 acces- sions of the HapMap collection. Red area is categorized as saline (>50% surface), and CaCO3 content (mg kg1) is shown (gray-scale legend). Frame shows the map of Catalunya, indicating the location of the saline calcareous soil (SCS) used in the experiment; (b) physico-chemical characterization of SCS. Ó 2022 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd., The Plant Journal, (2023), 113, 225–245 1365313x, 2023, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/tpj.16042 by Readcube (Labtiva Inc.), Wiley Online Library on [01/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 228 Maria Jose Almira Casellas et al. (a) (b) (c) (d) (e) (f) (g) Figure 2. Phenotypic analysis of the studied accessions grown on saline calcareous soil (SCS). (a) Scatter plot of the rosette diameter (cm) of all accessions grown on SCS. Each dot represents an accession. Growth categories were established by selecting accessions on the 10% top (blue) and 10% lowest (yellow) rosette diameter. HD: High Diameter group; LD: Low Diameter group; (b) Radial plot with the Z-values of the studied elements in leaves of HD and LD acces- sions. Axes display Z-scores calculated per element and asterisks show significant differences among the two extreme groups (Student t-Test, P < 0.05); (c) Chart pie of nutrient content in the native soil of HD (blue) and LD (red) accessions. Asterisks and histograms show the two elements displaying significant dif- ferences among the two groups (Student t-Test, P < 0.05). Plants were grown on SCS for 8 weeks (n = 4). (d) Pairwise correlation of leaf mineral nutrients and growth performance (Leaf Na and RD, leaf S and RD, expressed as lg g1 DW and cm, respectively); (e) Pairwise correlation of Leaf nutrient concentrations (Na and Fe, Fe and S, Fe and Mn, expressed as lg g1 DW); (f) Pairwise correlation of leaf mineral nutrients and SCS soil element concentrations (Leaf Na and soil Mg and Ca, expressed as lg g1 DW and g Kg1 respectively); (g) Pairwise correlation of SCS soil element concentrations (Ca and Mg, Fe and Mg, Ca and Mn, expressed as g Kg1). The lines represent the result of linear regression. R2: Squared Pearson correlation’s coefficient. Ó 2022 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd., The Plant Journal, (2023), 113, 225–245 1365313x, 2023, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/tpj.16042 by Readcube (Labtiva Inc.), Wiley Online Library on [01/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License GWAS on alkaline-salinity responses in thaliana 229 with rosette diameter (RD) (R2 = 0.05; R2 = 0.06). Results rest of leaf ion concentration phenotypes are provided in are represented in the correlogram, clustered heatmap and Figure S4. A single strong peak of SNPs associated with scatter plot matrix (Figure S3a–c). leaf Na+ at Chr 4, with the peak centered on AtHKT1. Previ- Next, leaf element concentrations were compared ous QTL mapping and GWAS identified QTLs for leaf Na+ among the established growth performance groups to fur- centered on AtHKT1 (Baxter et al., 2010; Rus et al., 2006). ther characterize element interactions under SCS and their Such genetic evidence supports that the peaks of SNPs effects on plant performance. Overall, accessions from both associated with leaf Na+ and Fe+ observed in our GWA LD and HD (worst and best performance, respectively) analysis represent true positive associations and not a showed remarkably different ionomic profiles when com- false positive caused by high degree of population struc- pared to each other. Individuals from LD had significantly ture present in A. thaliana (Atwell et al., 2010). To account lower Fe concentration and higher leaf Na concentration for a possible effect of the HKT1 locus on the identification than individuals from HD. In turn, HD accessions contained of the other loci identified in this GWAs, HKT1 allelic varia- significantly less Zn, S and Mo and more Ca than those from tions in HD and LD accessions were assessed. The strong LD (Figure 2b). Nutrient ratios with known biological signifi- HKT1 variant was confirmed for all accessions used in fur- cance in plant growth and nutrition were also assessed in ther analyses. Other significantly associated SNPs [log10 the two groups of accessions. LD plants showed signifi- (P) > 6.42; see Methodology] were identified on chromo- cantly higher Na:K, Mg:Ca, Zn:Fe, S:Fe ratios and signifi- somes (Chr) 1 (SNP 1) and 4 (SNP 2) for LNa, and on Chr 1 cantly lower Fe:Mn ratios than those from the HD group for LFe (SNP 3). A SNP for LNa at Chr 4 with LOD score (Figure S2). We confirmed that RD was negatively correlated 5.56 mapping 40 Kb upstream SNP 3 was included in the to leaf Na and S content and, in turn, that the negative corre- analyses (SNP 4) for being in moderate LD with SNP 3 lations between leaf Fe and leaf Na and S were endorsed. (R2 = 0.4), in order not to rule out any potential candidate, The leaf ionome is largely dependent on the soil nutri- as sub-threshold signals with proximity to putative candi- ent availability. It is well known that geographically wide- date genes have been previously selected to identify novel spread A. thaliana accessions have evolved mechanisms to loci involved in the studied response (Wang et al., 2016). balance their nutritional needs based on availability of The scores, positions, genes associated with the detected nutrients in the site of origin (Campos et al., 2021). Thus, SNPs and genes included in the linkage disequilibrium we further considered the correlations between the leaf and (LD) regions are detailed in Table S3. soil nutrients in the study accessions, and the relations The function and localization of all candidate genes among different soil nutrients at their natural habitats. Leaf included in the quantitative trait locus (QTL) regions was Na concentrations correlated positively with concentrations examined for all described peaks. This led to a total of 22 of Mg and Ca in the soil of origin. In turn, high soil Mg and candidate genes for analysis from which AT4G28900 was a Ca strongly correlated with high soil Fe and Mn, respec- transposable element and discarded. The other 21 genes tively (Figure 2g). were selected to identify the causal gene(s) underlying Finally, we analyzed the correlation between RD and leaf each QTL association to the observed phenotypes. elemental profile on SCS and the properties of their soil of Among the selected candidates three transporters were origin to further explore the possible relationship between found: one mitochondrial transporter, AT1G10680 (P- geographic regions and the leaf ionome under alkaline salin- glycoprotein 10, PGP10), one cytoplasmic vesicle transporter ity. Estimated soil Na content and other elemental profile AT1G10730 (Adaptor protein-1 mu-adaptin 1, AP1M1) and data of the native soils of the tested accessions were one Golgi to plasma membrane transporter, AT1G59820 extracted from public maps of the European Soil Data Centre (Aminophospholipid ATPase 3, ALA3). PGP10 encodes a P- (ESDAC) (Table S1). As shown, accessions from HD belong glycoprotein from the ABC Transporter Family. It associated to soils with more elevated Na and more limited Fe concen- with differential leaf Na accumulation together with AP1M1, trations than accessions from LD (Figure 2c). which encodes a protein belonging to the clathrin adaptor complex medium subunit family (Qiao et al., 2010). ALA3 Detection of SNPs associated with differential leaf encodes a transporter involved in Golgi to plasma membrane elemental profile on saline calcareous soil phospholipid transport (Poulsen, Lo  pez-Marques, McDowell, To identify genomic regions underlying the differential Okkeri, Licht, Schulz, & Palmgren, 2008) and associated with growth and ionomic responses to alkaline salinity, we con- differential leaf Fe accumulation. ducted GWA analysis on leaf elemental profiles of the Another four genes are regulating or involved in accessions grown for 8 weeks on SCS. We identified sev- kinase activity: one encoding a nuclear kinase inhibitor, eral significantly associated SNPs using a 25% FDR thresh- AT1G10690 (Siamese-Related 8, SMR8), one encoding a old calculated by the Benjamini-Hochberg procedure. From phosphoribosyl pyrophosphate synthase, AT1G10700 the 10 phenotypes analyzed, two traits gave significant (Phosphoribosyl pyrophosphate synthase 3, PRS3) and two associations: LNa and LFe (Figure 3). Manhattan plots for Casein-Kinase-I-like protein coding genes, AT4G28880 Ó 2022 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd., The Plant Journal, (2023), 113, 225–245 1365313x, 2023, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/tpj.16042 by Readcube (Labtiva Inc.), Wiley Online Library on [01/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 230 Maria Jose Almira Casellas et al. Figure 3. Genome wide association studies (GWAS) for leaf ionome traits of HapMap accessions grown on SCS. Manhattan plots displaying the GWAS results for (a) leaf Na concentration and (b) leaf Fe concentration in the studied accessions. The horizontal gray dash-line line corresponds to a nominal 0.05 signifi- cance threshold after Benjamini Hochberg (False Discovery Rate) correction. Numbers, red dots and arrows indicate the regions containing the significantly associated locus. Peak of SNPs centered on HKT1 is circled and labeled. Blue arrow indicates SNP included independently from SNP 3 in the analysis. x-axis: chromosomal position of SNP; y-axis: -log10(P-value). Plants were grown on SCS for 8 weeks (n = 4). (CKL3) and AT4G28860 (CKL4). All four genes associated region as YUC8 and NINJA, are AT4G28730 (GrXC5) and with differential leaf Na accumulation. SMR8 and PRS3 AT4G28850 (ATXTH26), respectively. GrXC5 encodes a mapped to Chr1. CKL3 and CKL4 mapped to chromosome Glutaredoxin Family Protein with glutathione disulfide oxi- 4 and both CKL proteins act as signal transductors mainly doreductase activity. It belongs to the Class I or “Classic” in the blue light signaling pathway (Tan et al., 2013). GRX’s, which are involved in stress response (Couturier Two genes associated with the two SNPs mapping on et al., 2011). Specifically, GrxC5 is present only in Brassi- Chr 4 for leaf Na accumulation are involved in phytohor- caceae and its dimeric holoprotein incorporates a [2Fe-2S] mone biosynthesis and signaling pathways: AT4G28720 cluster. ATXTH26 encodes a xyloglucan endotransglucosy- (YUC8) and AT4G28910 (NOVEL INTERACTOR OF JAZ, lase/hydrolase involved in root growth and cell wall exten- NINJA). YUC8 encodes a member of the flavin-binding sion (Maris et al., 2009). Another gene with a known monooxygenase family protein, which is involved in auxin biological function in this LD region is AT4G28750 (PSAE-1), biosynthesis and active in the nucleus. The gene is encoding a protein from the Photosystem I reaction center expressed mainly in flowers and shown to be ethylene subunit IV, involved in the response to light stimulus and responsive (Qin et al., 2017). NINJA encodes a transcrip- mRNA binding. tional repressor that functions in the jasmonic acid (JA) sig- Another gene with promising roles in nutrient home- naling pathway, has a role in root development, and a key ostasis present in the LD region of SNP 4 associated with role in leaf development (Pauwels et al., 2010). It is also differential leaf Fe concentration: AT1G59830 (Protein involved in the adaptive responses to salt stress (Ismail phosphatase 2a-1, PP2A-1). PP2A-1 encodes a serine/thre- et al., 2014). Two other genes included in the same LD onine phosphatase, which reverses the actions of protein Ó 2022 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd., The Plant Journal, (2023), 113, 225–245 1365313x, 2023, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/tpj.16042 by Readcube (Labtiva Inc.), Wiley Online Library on [01/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License GWAS on alkaline-salinity responses in thaliana 231 kinases. It is a regulator of stomatal development (Bian indicates the trait specificity of each locus association et al., 2020) and involved in the response to several abiotic except for AT4G28910 (NINJA) (Figure S5). stress factors such as heat (Wang et al., 2010), salt (Hu Effects of natural allelic variation of the locus of interest in et al., 2017), oxidative stress (Mathe et al., 2019) or drought gene expression and plant growth performance under (Rahikainen et al., 2016). alkaline salinity The other genes included in the analyses have roles in secondary metabolic processes (AT1G10690), cellular lipid To further identify the causal genes underlying SNP associ- metabolism (AT1G10720), homologous chromosome pairing ations, HapMap accessions showing extreme phenotypes at meiosis (AT1G10710), ubiquitin protein ligase binding (ExtP) for the studied traits were selected as “High Leaf (AT1G59800) or are described to encode nuclear proteins Na” (HLNa), “Low Leaf Na” (LLNa), “High Leaf Fe” (HLFe) (AT4G28890 and AT1G59810), with so far unknown func- or “Low Leaf Fe” (LLFe) accessions according to their leaf tions. Na and Fe contents, respectively (Table S5). Plants were To verify which of the candidate genes included in the re-examined for alkaline-salinity-induced changes in analyses were more likely to be responsible for the rosette size and ionomic profile under hydroponic condi- observed differential LNa and LFe observed under alkaline tions. The selected ExtP accessions were grown in control salinity conditions, we examined available homozygous and treatment solutions (0.5-strength Hoagland solution at T-DNA insertion mutant lines for each gene (Table S4). No pH 5.7 for control; 40 + 10 mM NaCl + NaHCO3 at pH 8.3 for seed viability or homozygous lines were obtained for the Alk-Sal treatment). Rosette diameter, root length, T-DNA insertion lines for AT1G10690 (SMR8), AT1G59810 rosette and root biomass and leaf ionome profiles were (AGL50), AT4G28750 (PSAE-1), AT4G28880 (CKL-3), assessed after 35 days. Allelic variation at the locus of AT1G10730 (AP1M1) and AT4G28890 (ATL42). A total of 12 interest was associated with differential growth (LD, HD) KO lines were analyzed. and ionomic responses (HLNa/LLNa; HLFe/LLFe) of the studied ExtP accessions. For the locus associated to differ- Mutant phenotyping ential LNa, the alternative variant correlated to the pheno- The 12 KO lines were grown together with Col-0 (wild type type LD and HLNa (sensitive accession with high leaf Na control) on SCS to perform the same phenotyping as con- accumulation) while the common variant correlated to HD, ducted with the HapMap studied accessions for the GWAS. LLNa (tolerant accession with low leaf Na accumulation) In parallel, a plate experiment was performed to observe (Figure 5a). For the locus associated to differential leaf Fe, severity of alkaline salinity effects on seed germination LD and LLFe phenotype (sensitive accession with low leaf and distinguish this from seed non viability (lines with no Fe accumulation) correlated to the common allele variant, germination neither in control nor in treatment conditions). while accessions bearing the alternative allele related to The effects of alkaline salinity conditions on seed germina- HD and HLFe groups (tolerant accession with high leaf Fe tion for all mutant lines and Col-0 and the performance of accumulation) (Figure 5b). each line on SCS are shown in Figure 4(a–c). All lines suf- We measured the expression of the candidate genes fered a dramatic inhibition in germination under alkaline in ExtP accessions under control and Alk-Sal hydroponic salinity conditions (Alk-Sal) (Figure 4a). For both SNPs with conditions. Accessions bearing the alternative and com- the highest associations with LNa on Chr 1 and 4 (SNPs 1 mon allele variants for each SNP were pooled separately. and 3, respectively, at positions 3 562 288 and 14 258 447), On the genes surrounding the LNa peak, PGP10 expression the Col-0 reference allele was associated with low LNa. was not significantly different based on this comparison From the candidate genes located in the respective LD (P = 0.89, Student t-test), while YUC8 and NINJA showed regions, pgp10 (Chr 1), yuc8 (Chr 4) and ninja (Chr 4) accu- significant differential expression in leaves between acces- mulated significantly higher leaf Na concentrations on SCS sions from group 1 (bearing the alternative allele) and (Figure 4b). For LFe, the Col-0 reference allele for SNP4 on group 2 (bearing the common allele) (P = 0.003, P = 0.015, chromosome 1 (located at position 22 010 370) associated P = 0.046; Student t-Test). Transcript levels of PGP10 did with low leaf Fe concentration. Significantly higher leaf Fe not correlate with plant growth (R2 < 0.001) but the relative was observed in ala3 when compared to Col-0 (Figure 4c). expressions of YUC8 and NINJA were highly correlated Growth constraints were observed in all selected lines with rosette diameter of each accession under Alk-Sal con- when compared to WT (Figure 4d). ditions (R2 = 0.55; R2 = 0.50 and R2 = 0.42, coefficient of The selected mutant lines for the candidate genes determination of the pairwise correlation) (Figure 6a). For mapping to differential LNa did not show significant alter- LFe peak, differences in ALA3 expression were not found ations in LFe when compared to Col-0 except for ninja, in shoots (Figure 6b) but roots (Figure 8b) (P = 0.0002) and which also displayed reduced LFe (P 0.034, Dunnett’s Test). gene expression levels correlated positively with plant per- Similarly, none of the selected mutants for LFe exhibited formance under Alk-Sal conditions (R2 = 0.45; R2 = 0.30, an altered LNa phenotype in comparison to WT. This respectively) (Figure 8b). Ó 2022 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd., The Plant Journal, (2023), 113, 225–245 1365313x, 2023, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/tpj.16042 by Readcube (Labtiva Inc.), Wiley Online Library on [01/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 232 Maria Jose Almira Casellas et al. (a) (b) 5000 ** 1.0 ** * Leaf Na ( µg /g DW) Germination Ratio 0.8 4000 0.6 0.4 3000 0.2 2000 0.0 1G l-0 1G 4 740 4G 0 ( 0 4G 20 1) AT 10 0 (c ) AT 10 0 ( 4) 59 0 ( -3) p ) 1G 1G 1) 28 0 ( 0 (g 3) ) 1G 86 c8 (p 0 C5 AT 071 885 4G 82 80 1000 AT 287 hs- 1G 70 kl- 0 p1 AT T 2A- 73 ala AT Co 1G 68 prs AT 28 (yu rx AT A 10 AT 59 59 83 pg 1 G2 p AT 710 ol-0 -)1 70 l-4) 1G 3) 4G 0 0 0 5) 8) 0 nja) ) 10 74 85 2 0 rs 28 grxc AT 28 yuc 07 T hs gp k (p A 10 28 i C 10 0 (n (c G1 (p ( (p 0 ( 0 0 AT 730 AT AT1 AT 86 72 1G 91 AT AT 68 10 28 28 10 1G 4G 4G 4G 4G 1G AT AT (c) (d) * 10 Rosette Diameter (cm) 150 Leaf Fe ( µg /g DW) 8 ** 100 6 ** ** 4 ** 50 2 0 ) ) 8) 3) ja 10 l-0 l-0 3) ) uc -1 la in gp ala Co (a Co 2A (n (y (p 0 0( 0 0 pp 82 72 91 0 82 68 59 0( 28 28 59 10 1G 83 4G 4G 1G 1G AT 59 AT AT AT AT 1G AT Figure 4. Phenotyping of T-DNA insertion mutants for the identified candidate genes. (a) Seed germination on plates under control and treatment conditions. Germination rate [(% Germination  Treatment)/(% Germination  Control)] of T-DNA and Col-0 lines. Plants were grown on agar plates for 1 week in control (½ MS at pH 5.9) or bicarbonate (½ MS at pH 8.3 and 10 mM NaHCO3) treatment conditions. n = 10 plants per accession and treatment. Mean  SE of leaf Na (b), and leaf Fe concentration (lg g1 DW) (c) and Rosette Diameter (cm) (d). Asterisks indicate significant differences between mutant and Col-0 wild type (*P < 0.05; **P < 0.01, Dunnet’s test). Plants were grown on SCS for 8 weeks (n = up to 6 plants per accession and treatment). Causal polymorphisms at the NINJA, YUC8 and ALA3 Three splice variants are associated to the NINJA locus locus. AT4G28910.1 and AT4G28910.2 are 2753 bp, while AT4G28910.3 is 207 bp shorter (2546 bp). The haplotypes After observing that allelic variation at the pinpointed candi- of the accessions with highest leaf Na – bearing the alter- dates is causal for the differential gene expression level and native allele of the marker SNP – and accessions with low- ultimately for the differential response to alkaline salinity in est leaf Na – possessing the non-reference allele at the the study accessions, the identification of most causal marker SNP - exhibit 5 SNPs: one in the 3’ UTR, 2 in the sequence polymorphisms was addressed by conducting hap- first intron for all 3 variants, 1 in the first intron of variant 1 lotype analysis on a 50-kb region centered on each candidate but within the splice region of variants 2 and 3, and one in gene. The pattern of polymorphisms of different haplotypes the first intron of variants 1 and 2 but within the splice that associated with contrasted phenotypes was compared region of variant 3. YUC8 is 1734 bp long with one exon. by the analysis of SNPs in intermediate (R2 > 0.6) and high Three SNPs in LD with the marker SNP surrounding the LD (R2 > 0.8) with the GWAS marker SNP. Ó 2022 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd., The Plant Journal, (2023), 113, 225–245 1365313x, 2023, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/tpj.16042 by Readcube (Labtiva Inc.), Wiley Online Library on [01/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License GWAS on alkaline-salinity responses in thaliana 233 (a) 0.8 200 ** * 180 Relative Leaf [Na] 0.7 Relative RD 160 0.6 140 120 0.5 100 0.4 80 0.3 60 Alternative Reference 40 Alternative Reference Allele Allele (b) 0.8 1.0 ** * Relative Leaf [Fe] 0.7 0.9 Relative RD 0.6 0.8 0.5 0.7 0.4 0.6 0.3 0.5 Alternative Reference Alternative Reference Allele Allele Figure 5. Phenotyping of selected accessions displaying extreme phenotypes and contrasted alleles for each SNP of interest under hydroponic conditions. From left to right, mean  SE of relative rosette diameter (Relative RD) per allele variant and relative leaf element concentration per allele variant for all LNa (a) and LFe (b) SNPs. Each dot represents 1 individual. Color boxes indicate advantageous (blue) and detrimental (yellow) alleles for each SNP. Plants treated with 0 or 40 mM NaCl + 10 mM NaHCO3 (pH 8.3) for 2 weeks. Three populations with up to 3 plants per population were pooled per each group. Asterisks indicate signifi- cant differences (*P < 0.05; **P < 0.01, Student t- Test). YUC8 coding region were found at the upstream, exon and comparing the two ExtP pools, while NINJA displayed sig- downstream YUC8 region. The exonic SNP is a synony- nificantly enhanced expression in the ExtP pool bearing mous variant. ALA3 is a 9083-bp gene. Haplotypes of the the alternative allele (P = 0.003, Figure 8a). HKT1 followed accessions with highest leaf Fe – bearing the reference the same trend and its expression level increased signifi- allele of the marker SNP – and accessions with lowest leaf cantly, but these differences were not caused by effects of Fe – possessing the alternative allele at the marker SNP - HKT1 allelic variation in the ExtP pools, as all used acces- exhibit 4 SNPs: 970 bp upstream the gene coding region, 2 sions were confirmed to carry the HKT1 reference (HLS, at the promoter region (639 and 529 bp upstream the cod- Col-0 -type) allele (Table S5). Root IRT1 and FRO2 did not ing region) and 1 at the third intron. show expression differences according to the study group SNP polymorphisms around the NINJA, YUC8 and classification, while ALA3 expression levels were dramati- ALA3 locus and SNPs differing from the Col-0 reference cally increased in ExtP accessions bearing the alternative genome sequence are shown in Figure 7(a–c). allele (P = 0.0003, Figure 8b). Identified novel loci are not masked by effects of known DISCUSSION major players in Na and Fe homeostasis Natural variation in the response of Arabidopsis thaliana To assess if expression patterns in the pinpointed candi- HapMap accessions to alkaline salinity date genes are determined by expression patterns of known major players in salinity and alkalinity tolerance, In this study, growth and nutritional status in a set of 270 the expression levels of HKT1 and SOS1 (for the leaf Na natural accessions of A. thaliana were screened under a SNP) and FRO2 and IRT1 (for the leaf Fe SNP) were ana- natural saline-carbonate soil and the molecular genetic lyzed in ExtP root samples. YUC8, NINJA (leaf Na SNP) mechanisms underlying stress responses to alkaline salin- and ALA3 (leaf Fe SNP) were included. SOS1 and YUC8 ity were explored by conducting GWAS. Among all acces- did not show differences in root expression levels when sions surviving to the end of the experiment, two groups Ó 2022 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd., The Plant Journal, (2023), 113, 225–245 1365313x, 2023, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/tpj.16042 by Readcube (Labtiva Inc.), Wiley Online Library on [01/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 234 Maria Jose Almira Casellas et al. (a) PGP10 YUC8 NINJA 5 1 ** 4 * HLNa 4 0.8 LLNa 3 3 Relative Gene Expression 0.6 2 2 0.4 1 1 0.2 0.0 0 0 2 8 R 2 = 7.25 e -5 R 2 = 0.550 R2 = 0.45 6 4 1.5 3 1 4 2 0.5 2 1 0 0 3 4 5 6 7 3 4 5 6 7 1 2 3 4 5 6 Rosette diameter (cm) (b) ALA3 LLFe 1.5 HLFe 1.0 Relative Gene Expression 0.5 0.0 0.4 R²: 0.018 0.3 0.2 0.1 0 1 2 3 4 5 6 7 8 Rosette diameter (cm) Figure 6. Effects of allelic variation of selected candidate genes in gene expression levels. From left to right, gene expression of PGP10, YUC8 and NINJA (a) and ALA3 (b) in selected natural populations displaying extreme LNa and LFe phenotypes and contrasted alleles (ExtP). Scatter plots of relative gene expression (y-axis) and plant rosette diameter (x-axis) are represented below each gene expression plot. The lines represent the result of linear regression. R2: Squared Pearson correlation’s coefficient. Expression levels were normalized to expression in Col-0. Error bars: SE Leaf and root samples of 3 populations with 3 plants per population were pooled per each group. Populations used are listed in Table S5. Data from three independent biological replicates each with two technical replicates are analyzed. Asterisks indicate significant differences (*P < 0.05; **P < 0.01, Student t- Test). Ó 2022 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd., The Plant Journal, (2023), 113, 225–245 1365313x, 2023, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/tpj.16042 by Readcube (Labtiva Inc.), Wiley Online Library on [01/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License GWAS on alkaline-salinity responses in thaliana 235 Figure 7. SNP polymorphisms surrounding the NINJA, YUC8 and ALA3 locus. Black triangles and orange lines: most likely candidate SNPs underlying the allelic effects. Gene orientation is indicated with an arrow on the right (a, c) and left (b). Exons are indicated with yellow boxes and introns with lines connecting them. Splice variants are shown when present. Reference and alternative allele nucleotydic changes and SNP position are marked below; chromosome positions are indicated at the top (picture from https://www.arabidopsis.org/index.jsp). were established: LD accessions (Low Diameter – plants high biodiversity in the mineral nutrient accumulation below the 10% quartile), with poor adaptation to SCS and known to exist among different populations within a spe- HD accessions (High Diameter – plants above the 90% cies reflecting the chemical composition, and the nutrient quartile), with good adaptation to SCS. Differences in leaf and trace element bioavailability from their native soils nutrition, allelic variants of final candidate genes and dif- (Baxter & Dilkes, 2012). The A. thaliana HapMap set of nat- ferential gene expression levels between HD and LD ural accessions comes from habitats with a broad range of groups under alkaline salinity are discussed. soil physicochemical parameters, including Na, Fe and CaCO3 concentrations. Using a geographically widely dis- Footprint of eco-geographical adaptation in the ionomic tributed collection of accessions offers the opportunity of profiles of A. thaliana natural populations identifying causal genetic variants important for local High natural variation in the ionomic profiles of the 270 A. adaptation, including those constituting adaptation to a thaliana accessions was observed, in accordance with the combined stress like alkaline salinity (Miller & Busch, 2021). Ó 2022 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd., The Plant Journal, (2023), 113, 225–245 1365313x, 2023, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/tpj.16042 by Readcube (Labtiva Inc.), Wiley Online Library on [01/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 236 Maria Jose Almira Casellas et al. (a) (b) Figure 8. Expression pattern of major players in salinity and iron deficiency in roots of ExtP accessions. Gene expression of NINJA, YUC8, HKT1 and SOS1 (a) and ALA3, IRT1 and FRO2 (b) in roots of ExtP accessions. Scatter plots of relative gene expression (y-axis) and plant rosette diameter (x-axis) are represented below ALA3 gene expression plot. The lines represent the result of linear regression. R2: Squared Pearson correlation’s coefficient. Expression levels were nor- malized to expression in Col-0. Error bars: SE Root samples of three accessions with three plants per accession were pooled per each group. Populations used are listed in Table S5. Data from three independent biological replicates each with two technical replicates are analyzed. Letters a and b indicate significant dif- ferences between mRNA expression levels (P < 0.05, HSD Tukey). We noticed that HD accessions originate from sites with saline soils in the study accessions, where not only a higher Na and lower Fe soil concentrations. As expected reduction in Na uptake or an enhancement in Na compart- for poorly adapted accessions, when grown on SCS LD mentalization (Volkmar et al., 1998), but also an efficient plants display higher leaf Na and lower leaf Fe concentra- acquisition of iron is advantageous (Li et al., 2016; Yousfi tions than HD plants. SCS study soil has an average EC of et al., 2007). Altogether it suggests that the enhanced 487 lS cm1, pH above 8 and an average CaCO3 concentra- response of HD plants to SCS is shaped by alkaline salinity tion of 19%. Thus, it is classified as moderately saline (Fan fitness promoting trade-offs. et al., 2011) and calcareous (de Santiago-Martin et al., HD accessions displayed lower Na:K under SCS when 2013; Teres, 2017). In arid and semiarid climates, high compared to LD individuals. It has been proven that alka- evapotranspiration rates move soluble Na+, Ca2+, Mg2+, K+, line salinity has a larger effect on the rice root Na:K ratio CO32 and HCO3 ions to the superficial soil layers leading than salinity alone (Shi & Sheng, 2005). This indicates that to the co-occurrence of alkaline-saline soils, which are esti- maintaining a low Na:K ratio in response to alkaline salin- mated to cover 831 million hectares of Earth (FAO, 1973). ity is a main mechanism of ion homeostasis regulation, as In this context, Fe uptake efficiency is critical to prevent it is for salinity alone (Hu et al., 2022). chlorosis under conditions of high soil pH (Hsieh & The HD group also showed significantly better Fe-S Waters, 2016) and salinity aggravates this problem by neg- adjustment, as the decreased leaf Fe accumulation in LD atively affecting the acquisition and internal use of Fe by accessions was accompanied by a strong increase in leaf S plants (Ben Abdallah et al., 2017; Rabhi et al., 2007; Zhou concentration. There is dynamic crosstalk between plant Fe et al., 2017). The observed results point to the presence of and S networks, which is reported to be critical under low a footprint of eco-geographical adaptation to alkaline Fe availability (Hantzis et al., 2018) but also under S Ó 2022 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd., The Plant Journal, (2023), 113, 225–245 1365313x, 2023, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/tpj.16042 by Readcube (Labtiva Inc.), Wiley Online Library on [01/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License GWAS on alkaline-salinity responses in thaliana 237 deficiency (Ostaszewska et al., 2014; Vigani & Briat, 2016). accumulation in bean (Cakmak, 2000) and, in the opposite Here, maintenance of Fe:S is favoring HD individuals under trend, iron deficiency led to Zn excess in maize (Akhtar SCS likely by optimizing Iron–sulfur (Fe–S) clusters, which et al., 2019). are at the center of photosynthesis, respiration, amino HD accessions displayed significantly higher Fe:Mn acid, and DNA metabolism (Mendoza-Co  zatl et al., 2019). ratio than LD accessions under SCS. Plants grown in SCS Iron–sulfur clusters are also required for most molybdo- showed Fe:Mn ratios ranging from 0.8 to 4. From the 12 enzymes to function (Bittner, 2014). HD accessions showed study accessions displaying Fe:Mn ratio below 1.5, 8 significantly lower leaf Mo accumulation. Molybdenum is a belonged to LD group. On the opposite trend, 15 out of 20 primary mineral nutrient highly available in alkaline soils accessions showing Fe:Mn ratio above 2 were from HD which can interfere with Fe acquisition when present in group. Classically, the optimum Fe:Mn ratio in plants has high concentrations (Alloway, 2013). Mo levels of the study been given as 1.5–2.5 (Shive, 1941). It is well established accessions on SCS (2–380 lg g1) are much larger than that Fe, Mn and other divalent ions share transporters (e.g. what was found for shoot Mo concentration ranges (0.7– IRT1) and that Fe-dependent chlorosis in A. thaliana can be 50 lg g1) in A. thaliana accessions grown in potting mix alleviated by means of sequestration of Mn in root vac- or hydroponics (Baxter et al., 2008). Thus, an adjustment in uoles (Eroglu et al., 2016). Thus, HD accessions might be Mo accumulation may favor, among others, Fe nutrition in more able to adjust Fe:Mn homeostasis by reducing Mn HD accessions, which in turn maintains Fe:S ratios and ulti- root-to-shoot translocation (Milner et al., 2013) or increas- mately Mo co-factor (Moco) biosynthetic activity (Teschner ing Mn import into root vacuoles (Li et al., 2019) leading to et al., 2010). increased Fe:Mn ratios under SCS. We observe significantly higher leaf Ca accumulation Detection of SNPs associated with differential leaf in HD accessions (Figure 2b). Interaction between salinity elemental profile on saline calcareous soil and Ca seems to play a pivotal role in the overall perfor- mance of the studied accessions: higher Ca concentrations In this study, we report significant natural variation of leaf under salinity inhibit Na+-induced K+ efflux by counteract- Na (LNa) and Fe (LFe) content in a panel of 270 Arabidop- ing the plasma membrane (PM) depolarization induced by sis natural populations exposed to saline carbonated soil. salt stress and thereby increase K+ absorption (Bacha We identified allelic variation in NINJA and YUC8 genes to et al., 2015; Shabala et al., 2006). In turn, high Ca levels in be likely causal for the variation in leaf sodium concentra- calcareous soils can alter Mg uptake and induce Mg defi- tion (LNa) and in ALA3 for the variation in leaf iron concen- ciency in plants (Mengel & Kirkby, 2001; Shaul, 2002), as tration (LFe). Mg and Ca are strongly antagonistic (Voogt 1988). Our Allelic variation in these genes leads to changes in results suggest that enhancing Ca nutrition - resulting in a their mRNA expression and is not associated to allelic vari- decrease in the Na:Ca ratio and an increase of K content –, ation in the main genes responsible for differential plant rather than maintaining low Ca:Mg ratios to avoid Mg defi- responses to either salinity (HKT1, SOS1) or alkalinity ciency, favors plant performance under alkaline salinity. (IRT1, FRO2). HD accessions accumulated significantly less Zn than Two out of the five SNPs detected in the NINJA gene LD accessions on SCS. Both high soil carbonate/bicarbon- are splice-site mutations - as they are found within splice ate and high Na affect Zn availability (Akhtar et al., 2019) sites (the boundary of an exon and an intron) in one or and our data show a significant negative correlation more NINJA splice variants. Both SNPs emerge as the top between RD and leaf Zn concentration (Figure S3b). How- candidates contributing to genotype-specific splicing ever, plants generally exhibit Zn deficiency symptoms at events under alkaline salinity at the NINJA locus, as alter- shoot concentrations below 15–20 lg g1 (Babuin native splicing (AS) regulates transcript levels (Grodecka et al., 2014) and HD accessions display higher average Zn et al., 2017) and contributes to plant adaptation to their concentration in leaf on SCS (73 lg g1). Moreover, Fe:Zn environment (Syed et al., 2012). One SNP in YUC8 causes ratios are significantly higher in HD accessions. Crosstalk an exonic synonymous variant. Far from having no effects in Zn and Fe nutrition has been extensively reviewed on the synthesized protein, coding synonymous variants (Pineau et al., 2012; Rai et al., 2021). Several Zn membrane can have a significant impact on mRNA structure, transla- transporters (mainly IRT-like proteins) are regulated by Fe tion rate and expression (Edwards et al., 2012). ALA3 con- status and can transport both Fe and Zn (Shanmugam tains 2 of the 4 assessed SNPs in the promoter region, et al., 2013; Vert et al., 2001; Yuan et al., 2008). Here, the which have a high potential to alter gene expression for enhanced Fe accumulation lead by alkalinity in HD plants being close to transcription start sites (Greenbaum & could be related to the observed decrease in leaf Zn con- Deng, 2017). The rest of potentially causal SNPs are in centration in these accessions and thus be a tolerance non-coding intragenic regions (Figure 8) and therefore they mechanism on SCS. These results are in line with previous do not have an obvious impact on gene expression or pro- studies in which Zn deficiency led to increased Fe tein conformation (Robert & Pelletier, 2018). Still, they Ó 2022 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd., The Plant Journal, (2023), 113, 225–245 1365313x, 2023, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/tpj.16042 by Readcube (Labtiva Inc.), Wiley Online Library on [01/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 238 Maria Jose Almira Casellas et al. might be good indicators for the leaf Na and Fe accumula- help to confirm its involvement in tolerance acquisition to tion phenotypes and for the expression level changes saline calcareousness in the soil and to elucidate their detected in the proximal genes. This suggests that YUC8, mechanism of action and possible mutual interaction. NINJA, and ALA3 could be genes under selection and AtALA3 encodes a phospholipid translocase active in might be affected by structural variation that should be fur- plasma membrane and the trans Golgi network. It has ther explored. been reported to be important for vegetative growth and NINJA (AT4G28910, Novel interactor of JAZ) acts as a reproductive success in A. thaliana. Screening of ala3 transcriptional repressor and is a negative regulator of jas- knockout mutants showed that ala3 was growth-defective monate responses. NINJA interaction with JAZ proteins dependent upon both temperature and soil when com- (jasmonate ZIM-domain repressor proteins) has been pared to Col-0 (McDowell et al., 2013). ALA3 contributes to experimentally determined. In turn, MYC2 interacts physi- secretory vesicle formation by assisting in the formation of cally with JAZ proteins, thus regulating JA-Ile-responsive slime vesicles in root tip cells (Poulsen, Lo  pez-Marques, gene expression. JA-Ile is a plant hormone that regulates a McDowell, Okkeri, Licht, Schulz, Pomorski, et al., 2008) and broad array of plant defense and developmental processes helps regulate polar growth by controlling phos- (Pauwels et al., 2010). Transcriptomic analysis has revealed phatidylserine exposure at the plasma membrane cytoplas- the importance of JA-Ile in the response of A. thaliana mic face (Lo  pez-Marques et al., 2021). Physical interaction plants to overcome moderate saline stress (Erice between AtALA3 and two ARFGEFs (ADPribosylation factor et al., 2017). GTPases) - BIG3 (Brefeldin A-Inhibited Guanine YUC8 (AT4G28720) is an auxin biosynthetic gene and Nucleotide-Exchange Protein3) and GNOM, which is essen- yuc8 mutants (null mutants of YUC8) have previously tial for proper trafficking of PIN auxin transporters – has been shown to suffer reduction in hypocotyl and petiole been observed in endomembrane, thus regulating traffick- length under both high and low red to far-red (R:FR) con- ing events in the late secretory pathway and ala3 mutants ditions. We showed that the yuc8 line developed signifi- are defective in auxin mediated developmental processes cantly smaller rosette, shorter roots and higher leaf Na (Zhang et al., 2020). There is increasing evidence for a role accumulation under alkaline salinity conditions when of auxin in plant response to both the Fe deficiency (Cel- compared to WT plants. It has been hypothesized that letti et al., 2020) and salinity (Cackett et al., 2022). Plant P4 down-regulation of auxin signaling might be part of A. ATPases are expected to have a major role in any physio- thaliana acclimation to neutral salinity (Iglesias et al., logical process involving lipid signaling, vesicle formation 2014). More recent studies have shown that exogenous and/or lipid-dependent protein recruitment to membranes. auxin application alters sodium ion accumulation and Plants sense environmental stimuli and transduce the sig- plays a protective role in salinity challenged strawberry nal into downstream biological responses often through (Zhang et al., 2021). Furthermore, a link between the plasma membrane, which is the interface between the enhanced auxin accumulation mediated by YUCCA genes cell and the environment and the source for signaling and a better developed root system architecture for the lipids. Salinity is well known to change membrane lipid acquisition of salinity stress tolerance has been hypothe- composition in plants, and to alter membrane permeability sized in rice (Saini et al., 2021). Several auxin biosynthetic for water, ions, and metabolites (Tsydendambaev genes have been reported as direct targets of YUC8: et al., 2013). Moreover, membrane lipids are directly AUF1, TAA1, TAR1, TAR2, shown to regulate local auxin affected by pH, due to their acid-basic properties. The biosynthesis in primary root tips (Ursache et al., 2014). assessment of the effects of an ALA3 hyper-functional The jasmonic acid signaling pathway has shown to be allele in membrane fluidity and permeability, regulation of linked to auxin homeostasis through the modulation of lipid-dependent signaling cascades and control of other YUC8, and roots of yuc8 and yuc9 knockout mutants dis- gene clusters that might activate plant adaptation under played a reduced response to methyl jasmonate (MeJA) alkaline salinity should be addressed using loss of func- (Hentrich et al., 2013). Phytohormones modulate a wide tion, homozygous knockout, and overexpressing mutant range of responses during the whole plant life cycle and lines for ALA3. the alteration of endogenous phytohormonal levels is a Altogether, we demonstrate Na-Fe homeostasis are key factor for the regulation of plant growth and reproduc- key features in the adaptation of A. thaliana to saline alka- tive responses to environmental stimuli. Here we suggest line conditions. Our GWAS approach using a saline cal- a role of YUC8 and NINJA expression levels in the regula- careous natural soil allowed the identification of new tion of auxin and jasmonic signaling pathways thus affect- players in this crucial ion homeostasis regulation: YUC8 ing plant tolerance to alkaline salinity. Quantification of and NINJA associated with Na leaf exclusion (providing a endogenous phytohormone concentrations and expression low leaf Na phenotype) and ALA3 with high Fe leaf accu- levels of direct targets of YUC8 and NINJA knockout and mulation. The importance of these genes for tolerance to overexpressing lines grown on alkaline salinity stress will saline alkaline conditions is further highlighted by the fact Ó 2022 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd., The Plant Journal, (2023), 113, 225–245 1365313x, 2023, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/tpj.16042 by Readcube (Labtiva Inc.), Wiley Online Library on [01/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License GWAS on alkaline-salinity responses in thaliana 239 that, excepting HKT-1, for which absence of allelic variation following the extraction method described by Busoms et al. (2018). in the study accessions has been confirmed, the study pop- Plant tissues were sampled by removing 2–3 leaves (1–5 mg dry weight) and washing them with 18 milli-Q water before being ulations bearing alternative and common allelic variants placed in Pyrex digestion tubes. Sampled plant material was dried for each identified locus do not differ in the expression of for 2 days at 60°C and weighed before open-air digestion in Pyrex well-stablished genes involved in Na and Fe homeostasis tubes using 0.7 ml concentrated HNO3 at 110°C for 5 h in a hot- such as SOS1, FRO1 and IRT1. Recent studies have shown block digestion system (SC154-54-Well Hot Block, Environmental that accessions with tolerance to moderate salinity regu- Express, Charleston, SC, USA). Concentrations of the selected ele- ments (Fe, Ca, K, Mg, S, Na, P, Mn, Mo, Cu, B, Zn and Ni) were late Na:K ratios under salinity by enhancing HKT1 expres- determined by ICP-MS (Perkin ElmerInc., ELAN 6000, MA, USA) or sion in roots but are unable to increase HKT1 root ICP-OES (Thermo Jarrell-Ash, model 61 E Polyscan, England). expression under alkaline salinity (Perez-Martın GWA studies and candidate genes selection et al., 2022). This suggests that mechanisms for a better maintenance of Na:K balance under salinity conditions are Genome Wide Association Studies (GWAS) was performed on the likely to differ from those under alkaline salinity and that phenotypes obtained from the 270 A. thaliana natural accessions the latter need to be better understood. that survived and grew on the SCS. Phenotypes included leaf min- eral concentrations of 11 elements (Fe, Mg, Ca, K, Na, S, Mn, B, P, Zn and Mo) of three 55-days old plants. Cu and Ni were below the EXPERIMENTAL PROCEDURES level of detection and could not be quantified. All the phenotypes are detailed in Table S1 and Figure S4. HapMap phenotyping and GIS data extrapolation GWA mapping was performed in GEMMA (version 0.98) Seeds of the Arabidopsis thaliana HapMap cohort (354 natural (Zhou & Stephens, 2012) with a univariate linear mixed model and accessions) were obtained from Nottingham A. thaliana Stock the minor allele frequency cut-off set at 0.1. GWAS was run with Centre (NASC, Nottingham, United Kingdom). The list and infor- the imputed 1 348 843 SNP set (Arabidopsis_2029_Maf001_ mation of the accessions is detailed in Dataset S1. Seeds were sur- Filter80) (Arouisse et al., 2020). A kinship matrix was constructed in face sterilized using 5% sodium hypochlorite (NaOCI) solution GEMMA to correct for population structure and individual related- under constant agitation for 10 min and rinsed with sterile water ness. The Benjamini & Hochberg threshold (BH) was implemented six times. Seeds were stratified for 4 days at 4°C after sowing to using the formula (i/m) Q, where: i = the individual P-value’s rank, synchronize germination, then four seeds per accession were m = total number of tests, Q = the false discovery rate set at 25%. sown in saline-carbonated soil (SCS) into Aratrays of 51 individual This corresponded to a BH threshold of 6.42. SNPs with score pots especially suited for A. thaliana (www.arasystem.com). The above BH correction and m.a.f > 0.1 were selected and the linkage SCS used for treatment was a mix of soil originating from a parcel disequilibrium (LD) region associated with the highest score SNP of natural saline-carbonated soil in L’Escala, NE Catalunya, Spain was explored to identify candidate genes for the causal allelic vari- (42°130 03″N 3°110 30″E) and perlite (3:1). The SCS had previously ant. All genes associated with the significant SNPs and their SNPs been characterized as suitable for saline-carbonate stress analysis in strong LD (r2 > 0.8) were explored (Table S3). Genes were anno- (Table S1) (Perez-Martın, 2020). Aratrays were placed in a growth tated according to TAIR 10 (Araport11). chamber with 150 lmol m2 sec1 of PAR, 10 h light/14 h dark Candidate genes validation photoperiod, and 25°C/20°C day/night temperature. Trays were bottom-watered twice weekly with deionized water with no further Seeds from 9 selected A. thaliana accessions, Col-0 and 17T-DNA nutrients added and aratrays were rotated horizontally to help insertion lines were obtained from the A. thaliana Stock Centre reduce gradient effects for light, temperature, and humidity. (NASC, Nottingham, United Kingdom). Each ExtP group (display- Germination and seedling survival were monitored. Fifty-five- ing contrasted phenotypes for leaf Na and Fe concentration and days old plants were harvested to measure their nutrient mineral bearing contrasted alleles at the candidate SNPs) was a pool of 3 contents and Rosette Diameter (RD), Rosette Leaf Area (RLA) and accessions. Figure S1 details the workflow and the A. thaliana Fresh Weight (FW) of three individuals per accession were mea- accessions and mutants used in each experiment. A. thaliana nat- sured at the end of the experiment. ural accessions were classified as “High Leaf Na” (HLNa), “Low To estimate the edaphic parameters of the native soil of each Leaf Na Lead” (LLNa), “High Leaf Fe Lead” (HLFe) or “Low Leaf A. thaliana accession, location coordinates and public maps from Fe” (LLFe) based on their leaf Na and Fe concentrations on SCS. the European Soil Data Centre (ESDAC) database (Panagos In all soil, hydroponic and plate experiments, seeds were surface et al., 2012) were combined using Q-GIS1. Natural accession coor- sterilized and stratified as described above to synchronize germi- dinates were extracted from GWAPP2 in WGS84 system (latitude nation. and longitude). Maps of soil properties at European scale, based on Lucas, 2009/2012 topsoil data from the European Soil Data Cen- Soil pot culture tre (ESDAC) were used to extract the following variables: pH (mea- Seeds were sown into the same soil type used for the HapMap sured in H2O), pH (in CaCl2 0.01 M solution), Cation Exchange screening. Each tray contained 24 individuals in a random block Capacity (CEC), Calcium carbonates (CaCO3), C:N ratio, and N, P, design with a total of six individuals per accession and soil type. and K concentrations (mg kg1). Plants were allowed to germinate and grow at 10 h light/14 h dark Elemental composition of soils and plants at 25°C to 20°C day/night temperature, 150 lmol m2 sec1 and 40% humidity. Trays were bottom watered twice weekly with To analyze the composition of the SCS, six independent samples deionized water. Rosette diameter (RD) from the third week to the from the trays used for the pot experiment (three at the beginning end of the experiment was measured from aerial pictures. Two and three at the end of the experiment) were collected. The soil leaves from 55-day-old plants were collected from up to six plants characterization was performed on the 2-mm fraction samples per accession and soil type. Ó 2022 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd., The Plant Journal, (2023), 113, 225–245 1365313x, 2023, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/tpj.16042 by Readcube (Labtiva Inc.), Wiley Online Library on [01/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 240 Maria Jose Almira Casellas et al. Hydroponic culture Statistical analyses Seeds were germinated in a mix of sand and perlite (3:1) and 10- All the statistical analyses were conducted using JMP SAS soft- day old seedlings were transferred to individual round hydroponic ware (SAS Institute, Cary, NC, USA). No data normalization was containers (100 ml) filled with 0.5-strength Hoagland solution (pH applied. To compensate for variation between and within trays, 5.7) and no NaCl or NaHCO3 added (control) or containing 40 mM outlier samples that were 4.5 times higher than the interquartile NaCl + 10 mM NaHCO3, set at pH 8.3 (treatment). Plants germi- range across all trays per each element were excluded from the nated and grew at 10 h light/14 h dark, 25°C to 20°C day/night tem- descriptive analysis. This threshold was chosen over the more perature, 150 lmol cm2 sec1 PAR and 40% humidity. The standard 1.5 times threshold according to Campos et al. (2021), to hydroponic solution was changed every 3 days to maintain a rela- include accessions with an extreme phenotype. tively constant concentration of nutrients in the solution and treat- In all soil and hydroponic cultures, mean-standardized values ment concentrations were increased two times - 20 mM ( 1 < value > 1) of elemental contents of soil and leaf material NaCl + 5 mM NaHCO3 at pH 8.3, and 30 mM NaCl + 15 mM NaHCO3 were used to represent the radar plots and compare between soil at pH 8.3 - until reaching the final concentration of 40 mM types, treatments or accessions, and One-way ANOVA was used to NaCl + 10 mM NaHCO3, at pH 8.3, when plants were 21 days old. test for significant differences (P < 0.05) between means of pheno- Plants remained at these conditions for two more weeks. 35-days typic responses, gene expression, and between means of elemen- old plants were harvested and root length, rosette diameter and tal contents of soil and leaf material. To perform multiple total fresh weight (biomass) of the six individuals per accession comparisons of group means we used Tukey’s HSD and to com- and per treatment were measured. Roots and leaves from the six pare elemental profiles of each mutant with Col-0 (WT) we used plants per accession were stored for ionome and gene expression Dunnett’s test. analysis. To investigate changes in root length (RL), rosette diameter (RD) and fresh weight (Biomass) in the hydroponic experiment, Plate culture where a control treatment was available, the phenotypic For germination assays, 10 seeds from each accession were responses to saline-carbonated conditions were quantified and sown in plates in a laminar air flow cabinet with sterile material. compared to the respective control conditions [relative measure- Plates were made for two treatments: control (½ MS at pH 5.9) ments: mean (XTreatment) = mean(XControl)]. and bicarbonate (½ MS at pH 8.3 and 10 mM NaHCO3). All plates Statistical data analyses are specified in Datasets S1–S5. contained Phyto-agar 0.6% (Duchefa, Haarlem, The Netherlands), and solutions were buffered with MES (2-(N-morpholino) ACCESSION NUMBERS ethanesulfonic acid) and BTP (Bis-Tris Propane) depending on Accessions numbers are listed in Supporting Information final pH. Plates with seeds were kept at 4°C for synchronizing germination. After 7 days under stratification treatment, plates Table S1. were moved to a growth chamber (12 h light/12 h dark, AUTHORS CONTRIBUTION 150 lmol m2 sec1, 40% humidity and 25°C). Germination was checked every day for 2 weeks and treatment effects were MJ-A and LP-M performed the lab experiments. MJ-A, RB recorded as (% Alk-Sal germination / % C-germination). Seeds of and SB performed data analysis. MJ-A, LP-M, ML and CP ninja and SALK_069095.55.25 mutant were not available at the time of plate experimental setup and were not assessed for ger- conceived the study. All authors contributed to the writing mination. of the manuscript. Gene expression analyses ACKNOWLEDGEMENTS Total RNA of about 100 mg of plant leaf material was extracted Special thanks to Rosa Padilla to process ICP field and soil sam- using the MaxwellR RSC plant RNA kit (Promega Corporation, ples. The authors are grateful to the Nottingham Arabidopsis Madison, WI, USA) following the manufacturer’s instructions. Stock Centre for the Arabidopsis seeds, to Dr. Ritter and Dr. Goos- Two micrograms of total RNA were used as a template to syn- sens, from the Specialized Metabolism Research Group at VIB- thesize first-strand cDNA with the iScriptTM cDNA Synthesis Kit UGent Center for Plant Systems Biology (Ghent University) for (Bio-Rad, Foster City, CA, USA). The cDNA was used as a tem- ninja knockout mutant seeds, and to Dr. Teres and Dr. Salt for plate for Reverse-Transcriptase quantitative real-time PCR (RT- sharing ionomic data for comparative purposes. This research qPCR) using iTaqTM Universal SYBR Green Supermix (Bio- was supported by the Spanish Ministry of Science and Innovation Rad). Real-time detection of fluorescence emission was per- (MICINN) (PID 2019 104000 RB I00). formed on a CFX384 Real-Time System (Bio-Rad), and plates CONFLICT OF INTEREST were edited using the CFX manager version 3.1 software. Pri- mers used for pinpointed candidate genes transcript quantifica- The authors declare no conflicts of interest. tion - PGP10, NINJA, YUC8, ALA3 – in selected natural accessions showing contrasted phenotypes are detailed in SUPPORTING INFORMATION Table S2. Relative quantifications were performed for all genes with the Actin2 gene (AT3G18780) used as an internal refer- Additional Supporting Information may be found in the online ver- ence. For each sample, the average value from triplicate RT- sion of this article. qPCRs was used to estimate transcript abundance. Four sam- Figure S1. Flowchart of the experimental procedure. Different ples per line were used. The mean Ct values were normalized color boxes define plant material used at each experimental stage: against Actin2 and dCt values were calculated as (dCtGene- Turquoise: HapMap cohort accessions; Purple: Col-0 (Wild-Type) dCtActin2). The data are expressed as means  SE relative to and T-DNA insertion mutant lines. the Col-0 value (defined as 1). Ó 2022 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd., The Plant Journal, (2023), 113, 225–245 1365313x, 2023, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/tpj.16042 by Readcube (Labtiva Inc.), Wiley Online Library on [01/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License GWAS on alkaline-salinity responses in thaliana 241 Figure S2. Nutrient ratios in leaves of the A. thaliana accessions Baxter, I., Brazelton, J.N., Yu, D., Huang, Y.S., Lahner, B., Yakubova, E. used in the study. et al. (2010) A coastal cline in sodium accumulation in Arabidopsis thali- ana is driven by natural variation of the sodium transporter AtHKT1; 1. Figure S3. Correlogram (a), clustered heatmap (b) and scatter plot PLoS Genetics, 6(11), e1001193. Available from: https://doi.org/10.1371/ matrix (c) of leaf element concentrations across the studied A. journal.pgen.1001193 thaliana accessions. Baxter, I. & Dilkes, B.P. (2012) Elemental profiles reflect plant adaptations to Figure S4. Genome wide association studies (GWAS) for leaf B, the environment. Science, 336(6089), 1661–1663. Available from: https:// Ca, K, Mg, Mn, P, S, Zn ionome traits of HapMap accessions doi.org/10.1126/science.1219992 grown on SCS. Baxter, I., Muthukumar, B., Park, H.C., Buchner, P., Lahner, B., Danku, J. et al. (2008) Variation in molybdenum content across broadly distributed Figure S5. SNP specificity of the identified candidate genes. populations of Arabidopsis thaliana is controlled by a mitochondrial Table S1. Leaf mineral nutrition, growth classification and native molybdenum transporter (MOT1). PLoS Genetics, 4(2), e1000004. Avail- soil characterization of the natural accessions collection used. able from: https://doi.org/10.1371/journal.pgen.1000004 Table S2. Primer set used for RT-qPCR expression analyses. Bazakos, C., Hanemian, M., Trontin, C., Jimenez-Gomez, J.M. & Loudet, O. (2017) New strategies and tools in quantitative genetics: how to go from Table S3. List for significant associations and corresponding QTLs the phenotype to the genotype. Annual Review of Plant Biology, 68, 435– detected by GWA mapping of Leaf Na and Fe concentration of 455. Available from: https://doi.org/10.1146/annurev-arplant-042916- plants grown on saline-calcareous study soil (SCS). 040820 Table S4. List for candidate gene T-DNA insertion mutant lines for  Ben Abdallah, H., Mai, H.J., Alvarez-Fern andez, A., Abadıa, J. & Bauer, P. leaf Na (LNa) and leaf Fe (LFe) phenotyping. (2017) Natural variation reveals contrasting abilities to cope with alkaline and saline soil among different Medicago truncatula genotypes. Plant Table S5. List for natural populations displaying extreme pheno- and Soil, 418(1), 45–60. Available from: https://doi.org/10.1007/s11104- types and contrasted alleles selected for growth, ionome and can- 017-3379-6 didate gene leaf and root expression analyses (ExtP accessions). Bian, C., Guo, X., Zhang, Y., Wang, L., Xu, T., DeLong, A. et al. (2020) Pro- Table S6. SNP polymorphisms surrounding the YUC8, NINJA and tein phosphatase 2A promotes stomatal development by stabilizing ALA3 locus in LD with the significant GWAS SNP. SPEECHLESS in Arabidopsis. Proceedings of the National Academy of Table S7. 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