The natural variation in shoot Na+ content and salt tolerance in maize is attributed to various minor‐effect variants, including an SNP located in the promoter of ZmHAK11

Exclusion of Na+ from the above-ground tissues serves as an important salt-tolerant mechanism in most glycophyte plants, such as maize (Munns and Tester, 2008). Existing studies have corroborated that different maize varieties exhibit significant diversity in shoot Na+ content and then salt tolerance (Liang et al., 2024). However, the genetic basis underlying this diversity remains largely unknown, necessitating a comprehensive understanding to sustain breeding for salt-tolerant maize cultivars. In recent decades, numerous genes have been identified to regulate Na+ transport. The well-known include genes from the NHX, HKT, HAK, CBL, and CIPK gene families (Yang and Guo, 2018). Maize has 13 NHX, 3 HKT, 28 HAK, 11 CBL, and 45 CIPK family members (Table S1), which exhibit a varied expression pattern and responses to salt treatment (Method S1; Figure 1a; Figure S1). Given that several genes within these families have been shown to underlie the diversity of shoot Na+ content and then salt tolerance in maize (Liang et al., 2024), we hypothesized that functional variation of additional members of these gene families may also do so. To substantiate this speculation, we obtained 14–623 SNP variants for each of these genes from the genotype data of a population comprised of 508 maize inbred lines (Zhang et al., 2019) (Table S1), then analyse the association between these SNP variants and Na+ content in the shoot tissue of salt-grown seedlings (Method S2). The result indicated that the peak SNP in 17 of these genes respectively explained >1% diversity of the shoot Na+ content (Figure 1b). Notably, 12 out of these 17 cases explained <2% diversity of the shoot Na+ content, supporting the notion that various minor-effect variants result in the diversity in the shoot Na+ content and salt tolerance in maize. The peak SNPs within ZmHAK4, ZmHKT1;1, ZmHKT1;2, and ZmHAK11 regions exhibited the highest contribution, respectively, explaining 7.5%, 4.4%, 4.0%, and 2.4% of the variation of the shoot Na+ content (Table S1). Considering ZmHAK4, ZmHKT1;1, and ZmHKT1;2 have been studied in previous studies (Liang et al., 2024), we determined the salt-tolerant role and functional variation of HAK11 in this study (Figure 1b). First, we created two independent knockout mutants, hak11-1 and hak11-2 (Method S3; Figure S2). Whilst the wild-type and hak11 plants did not show phenotypical differences under control conditions (Figure 1c), the shoot biomass of the mutants was around 20% smaller than the wild-type controls under salt conditions (Figure 1d). At the same time, the hak11 mutants exhibited a higher shoot and xylem sap Na+ concentration and lower root Na+ concentration than the wild-type under salt conditions (Figure 1e–g). These observations indicate that ZmHAK11 promotes shoot Na+ exclusion and salt tolerance by preventing root-to-shoot translocation of Na+. In alignment with this perspective, we observed that HAK11 unlikely to influence Na+ uptake or efflux in the root tissue (Figure S3) and the translocation of Na+ from shoot to root (Figure S4). The HAK family transporters were classified into four clusters (Cluster I–IV), with ZmHAK11 belonging to Cluster III (Figure S5). Existing reports have shown that Cluster I members (ZmHAK5 and ZmHAK1) and Cluster IV members (ZmHAK4 and ZmHAK17) are K+ and Na+ selective transporters, mediating response to K+ deficiency and salt stress, respectively (Qin et al., 2019; Zhang et al., 2019; Wang et al., 2024). ZmHAK11 was a plasma membrane-located protein (Figure S6). We found that ZmHAK11 increased the salt-sensitive phenotype of the yeast ant5 mutant, which is similar to the effect of the Na+ transporter ZmHAK4 (Method S4; Figure 1h). In contrast, ZmHAK5 (a K+ transporter) but not ZmHAK11 eliminated the growth impairment of yeast trk1 trk2 on a K+-deficient medium (Figure 1i). These results indicate that ZmHAK11 is capable of Na+ uptake, with undetectable K+ transport activity. Follow-up ion uptake assays supported this conclusion (Method S4; Figure j-m; Table S2–3), demonstrating that Na+-selective transporters can also be encoded by Cluster III of HAK family genes. Given that ZmHAK11 is preferentially expressed in the root tissue, including the root stele (Figure S7), we propose that ZmHAK11 probably promotes shoot Na+ exclusion by an intrinsic mechanism similar to that of HKT1 family transporters (Horie et al., 2009), that is, mediating the removal of Na+ from the root xylem flow. The peak SNP (Chr2_10505217) within the ZmHAK11 region is located in its seventh intron, at which guanine (G) and adenine (A) were associated with higher and lower Na+ content in the shoot tissue, respectively (Figure S8a,b). To examine the molecular basis underlying the functional diversity of ZmHAK11, we compared the ZmHAK11 transcript levels between 11 HapG and 11 HapA lines and found that salt stress significantly increased ZmHAK11 transcription in HapA lines but not in HapG lines (Figure 1n). Considering environmental-responsive gene expression is often linked to cis-regulatory elements within promoter region, we then cloned the ZmHAK11 promoter from the inbred line 3189 (a HapA line) and Ye8112 (a HapG line), generating pZmHAK113089-GFP and pZmHAK11Ye8112-GFP constructs. Subsequent assays observed that salt treatment significantly increased GFP transcription in tobacco leaves transformed with pZmHAK113089-GFP but not pZmHAK11Ye8112-GFP (Figure 1o). This indicates that variants in ZmHAK11 promoter confer the functional variation of ZmHAK11. To characterize the causal variant, we sequenced the ZmHAK11 promoter (around 2.0 kb) in 213 inbred lines and identified 191 SNP variants and 15 InDels (Table S4). The association between these variants and Na+ content was examined (Method S5), resulting in the identification of three significant SNP variants (SNP-1781, SNP-1437, and Chr2_10505217). Additionally, we found that both SNP-1437 and Chr2_10505217 exhibited moderate LD with the peak SNP (SNP-1781) (Figure 1p). The 213 maize inbred lines were categorized into four haplotypes based on the genotypes of these three significant SNPs: Hap1 (n = 170), Hap2 (n = 18), and Hap3 (n = 22) as the primary haplotypes, whilst Hap4 (n = 3) represented a minor haplotype that was excluded from subsequent assay (Figure 1q). Notably, we revealed that inbred lines carrying SNP-1781C (Hap2 and Hap3) exhibited increased ZmHAK11 transcript levels alongside reduced shoot Na+ content under salt conditions compared with those carrying SNP-1781G (Hap1) (Figure 1r,s). Moreover, we found that a C to G substitution (-1781C/G) significantly impaired the salt induction of pZmHAK113189 activity, to a level comparable to that of pZmHAK11Ye8112 (a SNP-1781G promoter) (Figure 1o), indicating that SNP-1781 is the variant that caused the functional variation of ZmHAK11. Moreover, we validated that the favourable allele of ZmHAK11 enhances the exclusion of Na+ from the shoot by using an F2 segregating population (Method S7; Figure 1t). In summary, we provide evidence that the diversity in shoot Na+ content and salt tolerance in maize is attributed to various minor-effect variants, including an SNP (SNP-1781) located in the ZmHAK11 promoter. In SNP-1781C lines, salt stress increases the transcript level of ZmHAK11, which then promotes the exclusion of Na+ from shoot tissue likely by mediating the removal of Na+ from the root xylem flow. In SNP-1781G lines, the salt induction of ZmHAK11 transcription is impaired, resulting in increased shoot Na+ content and sensitivity to salt stress. This study improves our understanding of the natural variation of maize salt tolerance and identifies new gene targets for the breeding of salt-tolerant maize cultivars. The authors acknowledge financial support from Biological Breeding-National Science and Technology Major Project (2023ZD04071), and the National Natural Science Foundation of China (32325037, 32201718, and 32401756). The authors declare no competing financial interests. X.L., L.W., P.Y., W.J., H.W., F.L., and C.J. conceived the research and wrote the manuscript. X.L. and L.W. performed the experiments. The data that support this study are openly available in China National Genomics Data Center at https://ngdc.cncb.ac.cn/search/all?&q=PRJCA016387. Figure S1–S8 Supplementary Figures. Method S1–S7 Supplementary Methods. Table S1–S3 Supplementary Tables. Table S3–S5 Supplementary Tables. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.