Loss of salt tolerance during tomato domestication conferred by variation in a Na + /K + transporter
2020; Springer Nature; Volume: 39; Issue: 10 Linguagem: Inglês
10.15252/embj.2019103256
ISSN1460-2075
AutoresZhen Wang, Yechun Hong, Guangtao Zhu, Yumei Li, Qingfeng Niu, Juanjuan Yao, Kai Hua, Jinjuan Bai, Yingfang Zhu, Huazhong Shi, Sanwen Huang, Jian‐Kang Zhu,
Tópico(s)Plant Molecular Biology Research
ResumoArticle5 March 2020free access Transparent process Loss of salt tolerance during tomato domestication conferred by variation in a Na+/K+ transporter Zhen Wang Shanghai Center for Plant Stress Biology and Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Yechun Hong Shanghai Center for Plant Stress Biology and Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China University of Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Guangtao Zhu Genome Analysis Laboratory of the Ministry of Agriculture, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen, China The AGISCAAS-YNNU Joint Academy of Potato Sciences, Yunnan Normal University, Kunming, China Search for more papers by this author Yumei Li The AGISCAAS-YNNU Joint Academy of Potato Sciences, Yunnan Normal University, Kunming, China Search for more papers by this author Qingfeng Niu Shanghai Center for Plant Stress Biology and Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Juanjuan Yao Shanghai Center for Plant Stress Biology and Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China University of Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Kai Hua Shanghai Center for Plant Stress Biology and Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Jinjuan Bai Shanghai Center for Plant Stress Biology and Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Yingfang Zhu Shanghai Center for Plant Stress Biology and Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China Collaborative Innovation Center of Crop Stress Biology, Institute of Plant Stress Biology, Henan University, Kaifeng, China Search for more papers by this author Huazhong Shi orcid.org/0000-0003-3817-9774 Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX, USA Search for more papers by this author Sanwen Huang Corresponding Author [email protected] orcid.org/0000-0002-8547-5309 Genome Analysis Laboratory of the Ministry of Agriculture, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen, China Search for more papers by this author Jian-Kang Zhu Corresponding Author [email protected] orcid.org/0000-0001-5134-731X Shanghai Center for Plant Stress Biology and Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN, USA Search for more papers by this author Zhen Wang Shanghai Center for Plant Stress Biology and Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Yechun Hong Shanghai Center for Plant Stress Biology and Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China University of Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Guangtao Zhu Genome Analysis Laboratory of the Ministry of Agriculture, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen, China The AGISCAAS-YNNU Joint Academy of Potato Sciences, Yunnan Normal University, Kunming, China Search for more papers by this author Yumei Li The AGISCAAS-YNNU Joint Academy of Potato Sciences, Yunnan Normal University, Kunming, China Search for more papers by this author Qingfeng Niu Shanghai Center for Plant Stress Biology and Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Juanjuan Yao Shanghai Center for Plant Stress Biology and Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China University of Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Kai Hua Shanghai Center for Plant Stress Biology and Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Jinjuan Bai Shanghai Center for Plant Stress Biology and Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Yingfang Zhu Shanghai Center for Plant Stress Biology and Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China Collaborative Innovation Center of Crop Stress Biology, Institute of Plant Stress Biology, Henan University, Kaifeng, China Search for more papers by this author Huazhong Shi orcid.org/0000-0003-3817-9774 Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX, USA Search for more papers by this author Sanwen Huang Corresponding Author [email protected] orcid.org/0000-0002-8547-5309 Genome Analysis Laboratory of the Ministry of Agriculture, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen, China Search for more papers by this author Jian-Kang Zhu Corresponding Author [email protected] orcid.org/0000-0001-5134-731X Shanghai Center for Plant Stress Biology and Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN, USA Search for more papers by this author Author Information Zhen Wang1,‡, Yechun Hong1,2,‡, Guangtao Zhu3,4,‡, Yumei Li4, Qingfeng Niu1, Juanjuan Yao1,2, Kai Hua1, Jinjuan Bai1, Yingfang Zhu1,5, Huazhong Shi6, Sanwen Huang *,3 and Jian-Kang Zhu *,1,7 1Shanghai Center for Plant Stress Biology and Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China 2University of Chinese Academy of Sciences, Shanghai, China 3Genome Analysis Laboratory of the Ministry of Agriculture, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen, China 4The AGISCAAS-YNNU Joint Academy of Potato Sciences, Yunnan Normal University, Kunming, China 5Collaborative Innovation Center of Crop Stress Biology, Institute of Plant Stress Biology, Henan University, Kaifeng, China 6Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX, USA 7Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN, USA ‡These authors contributed equally to this work *Corresponding author. Tel: +86 0755 2325 0159; E-mail: [email protected] *Corresponding author. Tel: +86 021 5707 8201; E-mail: [email protected] EMBO J (2020)39:e103256https://doi.org/10.15252/embj.2019103256 See also: Y Xiang & JM Jiménez-Gómez (May 2020) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Domestication has resulted in reduced salt tolerance in tomato. To identify the genetic components causing this deficiency, we performed a genome-wide association study (GWAS) for root Na+/K+ ratio in a population consisting of 369 tomato accessions with large natural variations. The most significant variations associated with root Na+/K+ ratio were identified within the gene SlHAK20 encoding a member of the clade IV HAK/KUP/KT transporters. We further found that SlHAK20 transports Na+ and K+ and regulates Na+ and K+ homeostasis under salt stress conditions. A variation in the coding sequence of SlHAK20 was found to be the causative variant associated with Na+/K+ ratio and confer salt tolerance in tomato. Knockout mutations in tomato SlHAK20 and the rice homologous genes resulted in hypersensitivity to salt stress. Together, our study uncovered a previously unknown molecular mechanism of salt tolerance responsible for the deficiency in salt tolerance in cultivated tomato varieties. Our findings provide critical information for molecular breeding to improve salt tolerance in tomato and other crops. Synopsis Selection of large fruits in domesticated tomato is linked to a reduction in salt tolerance. This study links domestication-associated variation in the Na+/K+ transporter-coding gene SlHAK20 to reduced salt tolerance in cultivated plants. Root Na+/K+ ratios show a strong positive correlation with fruit weight during tomato domestication. A variation in SlHAK20 is associated with Na+/K+ ratio modulation in tomato roots under salt stress conditions. The SlHAK20Hap1 haplotype is more effective than SlHAK20Hap2 in conferring Na+ homeostasis and salt tolerance. The role of SlHAK20 in regulation of salt tolerance is conserved in rice. Introduction Soil salinization is one of the major threats to crop productivity worldwide (Munns & Tester, 2008). As a predominant ion in saline soils, sodium (Na+) is absorbed from roots and accumulated in photosynthetic tissues, resulting in ionic imbalance, cellular toxicity, and thus reduced productivity. Plants possess a series of tolerance mechanisms to cope with salt stress, which includes limiting Na+ uptake, enhancing Na+ exclusion, adjusting cellular ionic balance (especially Na+/K+ ratio), and redistributing Na+ in leaves (Zhu, 2002, 2016; Ishikawa & Shabala, 2019). A number of membrane transporters are involved in Na+ and K+ influx and efflux processes and control the accumulation of Na+ and K+. Salinity-induced K+ transport and circulation in specific tissues lead to excess K+ accumulation that balances excess Na+ and thus confers salt tolerance in plants (Shabala & Cuin, 2008; Alvarez-Aragon et al, 2016; Wu et al, 2018). K+ could serve as a messenger for salt stress to inhibit energy-dependent biosynthesis processes but promote cell- and tissue-specific metabolism for the production of compounds in defense and repair of cellular systems during salt stress (Anschutz et al, 2014; Shabala & Pottosin, 2014; Shabala, 2019). K+ has even been considered as a determinant of cell fate by triggering programmed cell death via K+ leakage and generation of reactive oxygen species under salinity stress (Shabala, 2017; Rubio et al, 2019; Shabala et al, 2020). Identification and functional studies of these transporter genes regulating K+ and Na+ homeostasis will provide valuable resources for improvement of salt tolerance in crops. Ion homeostasis under salt stress is maintained by adjusting K+ and Na+ acquisition and distribution in plants (Wu et al, 2018; Isayenkov & Maathuis, 2019; Rubio et al, 2019). HKT1 (high-affinity potassium transporter 1) was first identified as a Na+/K+ symporter in wheat that contributes to salt tolerance by unloading Na+ from the transpiration stream (Schachtman & Schroeder, 1994), while its homologs, AtHKT1 and SKC1, were found to selectively transport Na+ in root cells and affect Na+ distribution between root and shoot (Berthomieu et al, 2003; Ren et al, 2005). The Na+/H+ antiporter SOS1 (Salt Overly Sensitive 1) is a plasma membrane transporter mediating Na+ extrusion and thus reducing cytosolic Na+ accumulation and toxicity. SOS1 was also suggested to load Na+ from parenchyma cells into xylem sap, whereas HKT1 mediates unloading of Na+ from xylem vessels to prevent Na+ overaccumulation in photosynthetic tissues (Shi et al, 2002; Berthomieu et al, 2003; An et al, 2017). The tonoplast-localized NHX-type Na+/H+ exchangers were reported to sequestrate Na+ into vacuoles but also play a role in K+ homeostasis (Blumwald, 2000; Bassil et al, 2011; Barragan et al, 2012). The HAK/KUP/KT (high-affinity K+/K+ uptake/K+ transporter) family transporters primarily mediate K+ fluxes, but some members of this family also play important roles in Na+ and Cs+ transport (Benito et al, 2012; Nieves-Cordones et al, 2017). In addition to maintaining K+ and Na+ homeostasis in plant tissues, these transporters are even involved in other cellular processes such as auxin movement and adenylate cyclase activation (Vicente-Agullo et al, 2004; Al-Younis et al, 2015). The HAK/KUP/KT transporters are classified into five major clades, clade I to clade V: Clade I is further divided into two (a and b), and the clade II is divided into three (a, b, and c) subclades (Nieves-Cordones et al, 2016). Among all the five clades, only three transporters in the clade IV have been characterized so far. LjKUP from Lotus japonicus complements K+ uptake deficiency in bacteria and responds to late nodulation development (Desbrosses et al, 2004). PhaHAK5 from salt-sensitive Phragmites australis shows Na+ permeability under high sodium stress (Takahashi et al, 2007). PpHAK13 from Physcomitrella patens is a high-affinity Na+ transporter with low K+ permeability, and its K+ transport capability is inhibited by high Na+ concentrations (Benito et al, 2012). GWAS is a powerful approach for uncovering the molecular basis of mineral composition in crops because more historical recombination, a larger number of alleles, and wider genetic variations can be investigated in an association study as compared to linkage analysis (Baxter et al, 2010; Chao et al, 2012; Yang et al, 2018; Kang et al, 2019). Tomato as a worldwide leading vegetable crop has publicly available sequences and an assembled genome (Sato et al, 2012; Lin et al, 2014). GWAS has been employed to resolve several traits in tomato, including fruit size, peel color, flavor, and metabolites (Lin et al, 2014; Tieman et al, 2017; Zhu et al, 2018). However, the genetic basis underlying natural variations in Na+ and K+ accumulation and salt tolerance in tomato remains to be identified. Here, we report a GWAS for root Na+/K+ ratio in a population with large natural variations. The most significant variation associated with root Na+/K+ ratio appeared to be within the gene SlHAK20 encoding a member of the clade IV HAK/KUP/KT transporters. We further revealed that SlHAK20 transports Na+ and K+ and regulates Na+/K+ balance under salt stress conditions. One major variant in the coding sequence of SlHAK20 was identified to be associated with Na+/K+ homeostasis and mediate salt tolerance in tomato. Knockout mutations in SlHAK20 in tomato and its homologous genes OsHAK4 and OsHAK17 in rice resulted in a sensitive phenotype to high salinity. Results Cultivated tomato varieties are substantially more sensitive to salt stress than wild ancestors To identify natural genetic variations associated with Na+ and K+ accumulation and salt tolerance in tomato germplasm, we measured Na+ and K+ contents in roots and shoots of 369 tomato accessions representing various geographical origins and improvement status. This population consists of 36 wild accessions of S. pimpinellifolium (PIM), 118 domesticated accessions of S. lycopersicum var. cerasiforme (CER), and 215 improved accessions of S. lycopersicum (BIG) (Figs 1A and EV1, Dataset EV1). We found that, after salt treatment for 1 day, the Na+ contents and Na+/K+ ratios in roots of the accessions in BIG group were significantly higher than those of the accessions in PIM and CER groups, whereas K+ contents in the accessions of the BIG group were notably lower than those in the accessions of PIM and CER groups. Both Na+ and K+ contents in roots showed no significant difference between PIM and CER groups, although the Na+/K+ ratios in the accessions of the CER group were comparatively higher than those in the PIM group. In shoots, no significant difference was identified among these three groups, although there was a higher Na+ content in the accessions of the BIG group than in the accessions of the CER group (Fig 1B, Table EV1). Interestingly, a strong positive correlation between fruit weight and root Na+/K+ ratio was found among the 195 accessions in these three groups (Fig 1C). This is coincided with the fact that tomato domestication and improvement have been focused on fruit appearance and yield rather than salt tolerance (Bolger et al, 2014; Tieman et al, 2017). Collectively, these data suggest that domestication and improvement processes for larger fruits in tomato have resulted in an increased ratio of Na+/K+ in roots under salt stress conditions. We further measured Na+/K+ ratio in the roots of four representative accessions including two PIM (TS-21 and TS-422) and two BIG (TS-577 and TS-670) accessions. The roots of TS-21 and TS-422 exhibited a lower Na+/K+ ratio than TS-577 and TS-670 (Fig 1D), which is consistent with the results obtained from the population. Since decreased Na+/K+ ratio is associated with increased salt tolerance (Munns & Tester, 2008), while vacuolar accumulation of Na+ resulting in higher Na+/K+ ratio has also been considered as a salt tolerance mechanism in plants (Wu et al, 2019), we thus evaluated the intracellular Na+ accumulation at the elongation zone of roots of these four accessions. Our results showed that TS-21 and TS-422 plants accumulated less cytosolic Na+ and were more resistant to high salinity than TS-577 and TS-670 plants (Fig 1E, Appendix Fig S1A). However, vacuolar accumulation of Na+ showed no significant difference among these four accessions (Appendix Fig S1B), suggesting that vacuolar Na+ sequestration is not the contributing factor for salt tolerance in these four contrasting accessions. Approximately 80% of the TS-21 and TS-422 plants survived the salt stress treatment as opposed to 30% of the TS-577 and TS-670 plants (Fig 1F), which supports a negative correlation between salt resistance and root Na+/K+ ratio in tomato. These results suggest that selection for large fruits in the process of domestication and improvement in tomato may be associated with a reduction in salt tolerance. Figure 1. Correlation of root Na+/K+ ratio with salt resistance during tomato domestication A. Distribution of 369 accessions, including PIM (36 S. pimpinellifolium accessions), CER (118 S. lycopersicum var. cerasiforme accessions), and BIG (215 S. lycopersicum accessions). B. The Na+ and K+ contents and Na+/K+ ratio in roots and shoots of three groups of 369 accessions after treatment with 150 mM NaCl for 1 day. C. Linear regression of root Na+/K+ ratio under salt stress (150 mM NaCl for 1 day) and fruit weight in 195 accessions from three groups. R2, coefficient of determination. n is the accession number of the three groups. D. The Na+/K+ ratios in roots of TS-21, TS-422, TS-577, and TS-670 after treatment with 150 mM NaCl for the indicated time (n = 3 biological repeats). E, F. Salt resistance assay of two PIM accessions (TS-21 and TS-422) and two BIG (TS-577 and TS-670). 20-day-old seedlings grown in soil were treated with 200 mM NaCl for 3 weeks (E). The survival rates were obtained from at least 12 plants in three repeated experiments (F). Data information: In (B), the box indicates the range of the percentiles of the total data determined using Tukey's method, the central line indicates the median, the whiskers indicate the interquartile range, and the outer dots are outliers. n indicates the number of accessions belonging to each group. The plots represent the means of three repeated experiments. Significant difference was determined by Student's t-test. In (F), the data are means ± SD (n = 3). Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Frequency distribution of Na+ and K+ contents and Na+/K+ ratio in root and shoot of 369 accessions A–C. Frequency distribution of Na+ (A), K+ (B), and Na+/K+ ratio (C) in roots of the tomato population. D–F. Frequency distribution of Na+ (D), K+ (E), and Na+/K+ ratio (F) in shoots of the tomato population. Data information: Red curve indicates the fitted line of the distribution. Download figure Download PowerPoint A variation in SlHAK20 is associated with root Na+/K+ ratio in tomato during salt stress Genome-wide association study has been successfully used to uncover the molecular basis of ion accumulation in plants (Baxter et al, 2010; Chao et al, 2012; Yang et al, 2018; Kang et al, 2019). However, the genetic basis underlying natural variations in Na+ and K+ accumulation and salt tolerance in tomato remains to be identified. To uncover the genetic alleles contributing to Na+ and K+ accumulation and salt tolerance, 2,824,130 common SNPs with a minor allele frequency (MAF) > 0.05 and missing ratio < 10% were used to perform a GWAS. The P-values of 1.0 × 10−7 were set as the significance threshold after Bonferroni-adjusted correction (Fig 2A, Appendix Fig S2). Nine major signals for root Na+/K+ ratio were detected with the most significant signal located on the short arm of chromosome 4 (Fig 2A). According to the linkage disequilibrium (LD) decay of genomic region harboring the most significant signal (Fig EV2), we retrieved the genes within 200 kb of the leading SNP (04_2156747) and considered their functions to select candidate genes responsible for root Na+/K+ ratio. A candidate locus, SlHAK20, was selected because it was annotated as a potassium transporter gene and the SNPs within this locus are close (86 kb upstream) to the leading SNP (Table EV2, Dataset EV2). This locus was also found to be in the regions associated with domestication sweep (Fig 2B, Dataset EV3), indicating that it underwent artificial selection for large fruits. Figure 2. Identification of SlHAK20 using GWAS of root Na+/K+ ratio A. Manhattan plot displaying the GWAS results of Na+/K+ ratio in root. The red dashed line indicates the Bonferroni-adjusted significance threshold (P = 1.0 × 10−7). Red arrow indicates the significant SNP signal of Na+/K+ ratio associated with SlHAK20. B. The nucleotide diversity ratios between PIM and CER, and between CER and BIG on chromosome 4. The black dashed horizontal lines indicate top 10% threshold for entire chromosome 4 (1.82 πPIM/πCER for domestication and 4.27 πCER/πBIG for improvement). The red arrows indicate the position of SlHAK20 in the sweeps. π, nucleotide diversity. C. SlHAK20-based association mapping and pairwise LD analysis. Dots represent SNPs. Indel 48 is highlighted in red. The indel −2,381, indel −1,818 in the promoter region, and five nonsynonymous variants are marked in blue. These eight variants are related to the pairwise LD diagram with dashed lines. D. Haplotypes of SlHAK20 among tomato natural variations. The distribution of Na+/K+ ratio in each haplotype group is exhibited by a box plot. n indicates the number of accessions belonging to each haplotype. In the box plots, the middle line indicates the median, the box indicates the range of the percentiles of the total data using Tukey's method, the whiskers indicate the interquartile range, and the outer dots are outliers. Significant difference was determined by Student's t-test. E. Allele distribution of the SlHAK20 locus at position in PIM, CER, and BIG groups. n indicates the accession number. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Average decay of linkage disequilibrium decay for the associated signal regionLinkage disequilibrium decay was estimated by squared correlation of allele frequencies (r2) against distance (Mb) among the SNPs. These SNPs were extracted from 2-Mb interval surrounding the leading SNP (ch04:2156747), which was determined by the ratios of Na+/K+ in root. Download figure Download PowerPoint The variations in the genomic sequence of SlHAK20 were identified among TS-21, TS-422, TS-577, and TS-670 based on the Heinz 1706 tomato genome assembly (version SL2.50) as a reference genome (Sato et al, 2012). In comparison, the coding region displays an in-frame 6 bp indel (named indel 48) variant significantly associated with root Na+/K+ ratio (P = 1.39 × 10−8), which is 48 bp downstream of the translation start codon (Dataset EV4). By resequencing of the SlHAK20 promoter regions from all accessions, two variants upstream of the ATG codon, an 48 bp indel (named indel −2,381) and a 32 bp indel (named indel −1,818), were identified but not associated with Na+/K+ ratio in root and shoot. The variant promoter designated as SlHAK20TS-670pro represents the promoters with deletions, and SlHAK20TS-21pro represents the promoters with insertions (Appendix Fig S3). In addition, other five nonsynonymous variations in the coding region show no significant effect on the phenotypic variation (Fig 2C, Table EV3). Based on the identified significant variations in SlHAK20, 369 accessions were classified into two haplotype groups. SlHAK20TS-21 belongs to Hap (haplotype group) 1 (n = 43), whereas SlHAK20TS-670 is the representative of the largest group Hap2 (n = 326), and these two contrasting accessions are used for further analysis in this study. Statistically, the accessions in Hap1 show significantly lower root Na+/K+ ratios than those in Hap2 (P = 6.258 × 10−8), and those in Hap1 show a lower shoot Na+/K+ ratio than those in Hap2. Therefore, we designated Hap1 as the tolerant alleles and Hap2 as the sensitive allele of SlHAK20 (Figs 2D and EV3A). We also measured the Na+ and K+ contents in the two haplotypes and found that Hap1 showed lower Na+ content in roots and shoots compared with Hap2 (Fig EV3B–E), suggesting that Hap1 is a stronger allele for restricting Na+ accumulation in a whole plant. The frequencies of the two alleles in PIM, CER, and BIG groups indicate that the salt resistance was lost progressively during domestication and improvement as larger fruits were selected (Fig 2E, Table EV4). These results indicate that the natural variation at the SlHAK20 locus is strongly associated with the difference in root Na+/K+ ratios among the accessions. Click here to expand this figure. Figure EV3. Analysis of Na+ and K+ contents in two SlHAK20 haplotypes in the population A–E. Haplotype analysis of the SlHAK20 gene based on shoot Na+/K+ ratios (A), root Na+ (B) and K+ (C), and shoot Na+ (D) and K+ (E) contents. Box plots represent the interquartile range using Tukey's method, the line in the middle of each box represents the median, the whiskers represent the interquartile range, and the dots represent outlier points. n indicates the number of accessions belonging to each haplotype. Significant difference was determined by Student's t-test. Download figure Download PowerPoint SlHAK20 functions in transport of Na+ and K+ As one of the two members in the clade IV of KT/KUP/HAK family, SlHAK20 homologous proteins have been identified from other crops (Nieves-Cordones et al, 2016), but no homologs were found in Arabidopsis (Fig 3A, Tables EV5 and EV6). Promoter–GUS analysis showed that the GUS activity driven by the two types of promoters was clearly stronger in shoots than in roots and high expression was detected in vascular tissues (Fig 3B, Appendix Fig S4). In the cross-sections of hypocotyl and root, GUS activity was primarily detected in the parenchyma cells surrounding xylem vessels (Fig 3C and D). The YFP fusion proteins of SlHAK20Hap1-YFP and SlHAK20Hap2-YFP exhibited exclusive localization in the plasma membrane (Fig 3E), indicating that the variations in the coding region have no effect on the subcellular localization of the proteins. The transport activity of SlHAK20 variants for K+ and Na+ was tested in the auxotrophic yeast mutants R5421 and ANT3, respectively (Shi et al, 2002; Li et al, 2014). Under K+-deficient conditions, the growth of R5421 was significantly depressed, while the SlHAK20 variants SlHAK20Hap1 and SlHAK20Hap2 could partially rescue the growth defect of R5421 mutant (Fig 3F). This result indicates that SlHAK20 mediates K+ influx transport. When grown in the presence of external Na+, the yeast cells expressing SlHAK20Hap1 exhibited higher Na+ transport activity than the yeast cells expressing SlHAK20Hap2 (Fig 3G). This result indicates that SlHAK20 also transports Na+ and the variation in SlHAK20 alters Na+ transport activity. Ion uptake kinetic analysis showed that SlHAK20Hap1 had a lower Km (26.8 ± 4.4 μM) than SlHAK20Hap2 (Km = 40.1 ± 4.7 μM), but comparable Vmax (76.5 ± 3.9 nmol/107 cells/h for SlHAK20Hap1 and 74.1 ± 3.2 nmol/107 cells/h for SlHAK20Hap2) in transporting Na+, indicating that SlHAK20Hap1 has higher Na+-binding affinity than SlHAK20Hap2 (Fig 3H). Taken together, our results support that Hap1 is a more active allele of SlHAK20 and confers salt tolerance by reducing Na+/K+ ratio in roots. Figure 3. Functional characterization of SlHAK20 A. Phylogenetic tree of the HAK/KUP/KT transporter family clade IV. The phylogenetic tree was constructed based on the amino acid sequences using the neighbor-joining method with MEGA5 in dicots and monocots. B–D. The expression pattern of SlHAK20 in tomato. The GUS activity was examined in seedling (left) and root (right) of 21-day-old SlHAK20TS-21pro:GUS transgenic tomato (B). Scale bars, 2 cm. Cross-sections of hypocotyl (C) and root (D) of SlHAK20TS-21pro:GUS transgenic plants. The scale bar is 400 μm in (C) and 200 μm in (D). E. Subcellular localization of SlHAK20-YFP in tomato. The SlHAK20Hap1-YFP (left) and SlHAK20Hap2-YFP (right) were localized in the plasma membrane. YFP, yellow fluorescence protein. FM4-64, a lipophilic styryl compound as a red fluorescent marker of plasma membrane. Scale bar, 20 μm. F. SlHAK20 complements the K+ uptake-defective yeast muta
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