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Variation in TaSPL6-D confers salinity tolerance in bread wheat by activating TaHKT1;5-D while preserving yield-related traits

Nature Genetics (2024)Cite this article

Abstract

Na+ exclusion from above-ground tissues via the Na+-selective transporter HKT1;5 is a major salt-tolerance mechanism in crops. Using the expression genome-wide association study and yeast-one-hybrid screening, we identified TaSPL6-D, a transcriptional suppressor of TaHKT1;5-D in bread wheat. SPL6 also targeted HKT1;5 in rice and Brachypodium. A 47-bp insertion in the first exon of TaSPL6-D resulted in a truncated peptide, TaSPL6-DIn, disrupting TaHKT1;5-D repression exhibited by TaSPL6-DDel. Overexpressing TaSPL6-DDel, but not TaSPL6-DIn, led to inhibited TaHKT1;5-D expression and increased salt sensitivity. Knockout of TaSPL6-DDel in two wheat genotypes enhanced salinity tolerance, which was attenuated by a further TaHKT1;5-D knockdown. Spike development was preserved in Taspl6-dd mutants but not in Taspl6-aabbdd mutants. TaSPL6-DIn was mainly present in landraces, and molecular-assisted introduction of TaSPL6-DIn from a landrace into a leading wheat cultivar successfully improved yield on saline soils. The SPL6-HKT1;5 module offers a target for the molecular breeding of salt-tolerant crops.
通过 Na + -选择性转运蛋白 HKT1;5 将 Na + 从地上组织排除是作物的主要耐盐机制。通过表达全基因组关联研究和酵母单杂交筛选，我们鉴定了 TaSPL6-D，它是面包小麦中 TaHKT1;5-D 的转录抑制因子。 SPL6 还针对水稻和短柄草属中的 HKT1;5。 TaSPL6-D 第一个外显子中的 47-bp 插入导致截短的肽 TaSPL6-D In ，破坏 TaSPL6-D Del 表现出的 TaHKT1;5-D 抑制。过表达 TaSPL6-D Del 而不是 TaSPL6-D In 会导致 TaHKT1;5-D 表达受到抑制并增加盐敏感性。两种小麦基因型中 TaSPL6-D Del 的敲除增强了耐盐性，而进一步敲除 TaHKT1;5-D 则减弱了耐盐性。在Taspl6-dd突变体中保留了尖峰发育，但在Taspl6-aabbdd突变体中则不然。 TaSPL6-D In 主要存在于地方品种中，通过分子辅助将 TaSPL6-D In 从地方品种引入到主要小麦品种中，成功提高了盐渍土的产量。 SPL6-HKT1;5 模块为耐盐作物的分子育种提供了目标。

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Fig. 1: The identification of TaSPL6-D as an upstream regulator of TaHKT1;5-D via a combined strategy of Y1H screening and eGWAS.
Fig. 2: TaSPL6-DDel can bind to and negatively regulate TaHKT1;5-D, whereas TaSPL6-DIn cannot.
Fig. 3: TaSPL6-DDel attenuates salinity-stress tolerance of bread wheat.
Fig. 4: Knockdown of TaHKT1;5-D in Taspl6-dd mutant subdues the salinity-tolerant phenotype.
Fig. 5: Four genes encoding activators of TaHKT1;5-D are also downstream targets of TaSPL6-D.
Fig. 6: The homoeologous redundancy of bread wheat ensures normal spike development in Taspl6-dd.
Fig. 7: TaSPL6-DIn represents an elite allele not fully used in modern breeding, and its introgression from landraces into modern cultivars enhances salinity tolerance on saline farmlands.

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Data availability

The raw data files from the CUT&Tag-seq have been deposited in the Gene Expression Omnibus database with publicly available accession GSE245464. The initial mutation information of EMS mutants was collected from the Wheat JING411 TILLING Database (http://jing411.molbreeding.com) and WheatOmics v1.0 (http://wheatomics.sdau.edu.cn). Other data supporting the findings of this work are available within the paper and its Supplementary Information files. Source data are provided with this paper.

Code availability

All software used in this study is publicly available on the Internet, as described in the Methods and Reporting Summary.

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Acknowledgements

We appreciate H. Li (State Key Laboratory of Crop Stress Adaptation and Improvement, College of Agriculture, Henan University) for helping us obtain the haplotype information of SPL6 in the A. tauschii population. We also appreciate M. Zhang (China Agricultural University) for technical assistance in the isolation of root stele cells; G. Li (Nanjing Agriculture University) and Y. He (Jiangsu Academy of Agricultural Sciences) for technical assistance in the immunoblotting assay; S. Liu (Nanjing Agriculture University) for leaf extraction from images of field trials. This work was supported by the National Natural Science Foundation of China (U1906202 and 32072064), the National Key Research and Development Program of China (2022YFD1900704-7), the Natural Science Foundation of Jiangsu Province, China (BK20200110), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA24020104) and Youth Innovation Promotion Association of Chinese Academy of Sciences (2022314).

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  1. These authors contributed equally: Meng Wang, Jie Cheng, Jianhui Wu.

Authors and Affiliations

  1. State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing, P. R. China

    Meng Wang, Jie Cheng, Jiefei Chen, Dan Liu, Chenyang Wang, Guangjie Li, Dongwei Di & Weiming Shi

  2. University of Chinese Academy of Sciences, Beijing, P. R. China

    Meng Wang, Jiefei Chen & Weiming Shi

  3. State Key Laboratory of Crop Stress Resistance and High-Efficiency Production, College of Agronomy, Northwest A&F University, Yangling, P. R. China

    Jie Cheng, Jianhui Wu, Dan Liu, Chenyang Wang & Dejun Han

  4. State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, P. R. China

    Shengwei Ma

  5. Hainan Yazhou Bay Seed Laboratory, Sanya, P. R. China

    Shengwei Ma

  6. Shandong Engineering Research Center of Germplasm Innovation and Utilization of Salt-Tolerant Crops, College of Agronomy, Qingdao Agricultural University, Qingdao, P. R. China

    Weiwei Guo & Yumei Zhang

  7. School of BioSciences, Faculty of Science, The University of Melbourne, Parkville, Victoria, Australia

    Herbert J. Kronzucker

  8. The Key Laboratory of Plant Development and Environment Adaptation Biology, Ministry of Education, School of Life Science, Shandong University, Qingdao, P. R. China

    Guangmin Xia

  9. International Research Centre for Environmental Membrane Biology, Foshan University, Foshan, P. R. China

    Weiming Shi

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Contributions

M.W. conceived and supervised the project, performed most of the experiments, analyzed the data and wrote the manuscript. J.W. managed the bread wheat natural population, performed eGWAS and haplotype analysis and wrote the corresponding sections in the manuscript. J. Cheng performed most of the experiments related to the function of TaSPL6. J. Chen conducted some phenotypic assays related to salt stress. D.L. conducted some phenotypic assays related to spike development. C.W., W.G. and Y.Z. contributed to the field trials. S.M. provided assistance in eGWAS. G.L., D.D. and D.H. discussed the manuscript. H.J.K., G.X. and W.S. discussed and revised the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Meng Wang.

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Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Genetics thanks Tomoaki Horie, Caifu Jiang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Transcriptional analysis of bread wheat TaHKT1;5-D to determine the optimal sample for Y1H screening and eGWAS.

(a) Schematic of the isolation of root-stelar samples. Scale bar, 1 cm (left panel); 25 μm (the rest three panels). (b) The relative expression level of TaHAK4-7A (TraesCS7A02G071000) and (c) TaHKT1;5-D (TraesCS4D02G361300) in the stele or outer part of roots. TaHAK4-7A was used as a positive control for the stele-specific expression. TaEF1-α (M90077) was used as the endogenous control. The transcript abundance in the outer part of the roots was normalized to 1 in each panel. Each bar represents the mean ± SD (n = 3 independent experiments). Significance analysis was performed by two-tailed Student’s t test. (d) Global analysis of the transcriptional response of TaHKT1;5-D in root-stelar cells of 22 representative bread wheat accessions to salt stress. (e) Subgroup analysis of the transcriptional response of TaHKT1;5-D in root-stelar cells to salt stress. Twenty-two representative bread wheat accessions were divided into two subgroups, TaSPL6-DIn accessions (n = 8) and TaSPL6-DDel accessions (n = 14), by the core 47-bp InDel variation. In the box plots, centerline represents median and bounds of box indicate the interquartile range. Each bar represents the mean ± SE. h, hour; D, day.

Source data

Extended Data Fig. 2 Genetic structure of the 304 included wheat accessions and eGWAS results for the expression level of TaHKT1;5-D.

(a) The distribution of filtered SNPs/InDels across the wheat genome. (b) Neighbor-joining tree analysis, (c) structure analysis and (d) principle component analysis to reveal the population structure. Vertical lines in c indicate genetic similarity thresholds used to classify accessions into two main groups. (e) Results of eGWAS for the expression level of TaHKT1;5-D. Significance was calculated by two-tailed Fisher’s exact test. The black horizontal dashed line indicates the genome-wide suggestive significance threshold (P = 1.18 × 10−6). The red horizontal dashed line indicates the significance threshold (P = 1.00 × 10−7) to strictly define significant loci associated with the expression level of TaHKT1;5-D. (f) Quantile–quantile plot for the GWAS under the mixed linear model (MLM). The gray layer indicates the error band (±5%). (g) Nucleotide diversity across the gene TaSPL6-D. A 1-bp sliding window with a 1-bp step size was used to calculate nucleotide diversity (π). The core InDel variation (iad5D_540783796) is highlighted by arrows.

Source data

Extended Data Fig. 3 The transcription profiles of TaSPL6 homoeologs.

(a) Spatial expression patterns of TaSPL6 homoeologs. (b) Transcription of TaSPL6 homoeologs in the root stele under control (CT) and salt-stress conditions. TaEF1-α (M90077) was used as the endogenous control. Each bar represents the mean ± SD (n = 3 independent experiments). Significance analysis was performed by one-way LSD test.

Source data

Extended Data Fig. 4 Dual-luciferase transient expression assay using the TaHKT1;5-D promoter harboring the wild-type or mutated GTAC motifs.

(a) Schematic representation of various constructs used in the dual-luciferase (LUC) transient expression assay. (b,c) Dual-luciferase transient expression assay shows that all three GTAC motifs contributed to the suppression of the promoter activity of TaHKT1;5-D by TaSPL6-DDel. Scale bar in b, 1 cm. Each bar in c represents the mean ± SD (n = 8 biologically independent samples). Significance analysis was performed by two-tailed Student’s t test. EV, empty vector. (d) Immunoblot assay using anti-GFP antibody showing TaSPL6-DDel abundances in b and c. Tubulin served as the loading control. Molecular masses in kD are indicated on the left side. For cropped blots, the samples derived from the same experiment and blots were processed in parallel. Experiments were independently performed at least three times with similar results.

Source data

Extended Data Fig. 5 SPL6 in rice and Brachypodium distachyon can target and suppress HKT1;5.

(a) Position and conservation of the GTAC motifs present in the promoters of TaHKT1;5-D, OsHKT1;5, HvHKT1;5 and BdHKT1;5. (b) Y1H assay results. SD/−Leu, SD medium without Leu; SD/−Leu/AbAX, SD medium without Leu supplemented with AbA at the concentration of 100 or 200 ng mL−1. Transformed yeast cells were dotted at 10−1 dilutions on the selective medium. Experiments were independently performed at least three times with similar results. (c,d) Schematic representation of various constructs used in the dual-luciferase (LUC) transient expression assay. (e,f) Dual-luciferase transient expression assay showing that OsSPL6 or BdSPL6 is able to suppress the promoter activity of OsHKT1;5 or BdHKT1;5, which was related to GTAC motif. EV, empty vector. Scale bar in e, 1 cm. Each bar in f represents the mean ± SD (n = 8 biologically independent samples). Significance analysis was performed by two-tailed Student’s t test. (g) Immunoblot assay using anti-GFP antibody showing OsSPL6 and BdSPL6 abundances. Tubulin served as the loading control. Molecular masses in kDa are indicated on the left side. For cropped blots, the samples derived from the same experiment and blots were processed in parallel. Experiments were independently performed at least three times with similar results.

Source data

Extended Data Fig. 6 EMS-based mutants of TaSPL6-DDel in the background of bread wheat cv. JING411 also enhances salinity tolerance.

(a) Mutagenesis of TaSPL6-DDel in cv. JING411. Green boxes and black line represent exons and introns, respectively. (b) Phenotypic performance, (d) shoot Na+ content and (e) whole-seedling biomass of wild-type (WT), TaSPL6-D-m1 and TaSPL6-D-m2 lines under control (CT) or salinity-stress (150 mM NaCl for at least 10 days) conditions. Scale bar in b, 4 cm. Each bar in represents the mean ± SD (n = 6 biologically independent samples) in d and (n = 7 biologically independent samples) in e. (c) The relative expression level of TaHKT1;5-D in root-stelar cells of WT, TaSPL6-D-m1 and TaSPL6-D-m2 lines under CT or salinity-stress (150 mM NaCl for 24 h) conditions. Transcript abundance in WT was calculated by giving the value 1. TaEF1-α (M90077) was used as the endogenous control. Each bar represents the mean ± SD (n = 3 independent experiments). In c, d and e, significance analysis was performed by one-way LSD test.

Source data

Extended Data Fig. 7 EMS-based mutants of TtSPL6-A in the background of tetraploid wheat cv. Kronos shows similar salinity tolerance to wild-type plants.

(a) Mutagenesis of TtSPL6-A and TtSPL6-B in tetraploid wheat cv. Kronos. (b) Phenotypic performance, (c) shoot Na+ content and (d) whole-seedling biomass of wild-type (WT), Ttspl6-aa-1 and Ttspl6-aa-2 lines under control (CT) or salinity-stress (100 mM NaCl for at least 10 days) conditions. Scale bar in b, 4 cm. Each bar in represents the mean ± SD (n = 6 biologically independent samples) in c and (n = 7 biologically independent samples) in d. In c and d, significance analysis was performed by one-way LSD test.

Source data

Extended Data Fig. 8 Four transcription factors encoded by TaSPL6-D targets can target and activate TaHKT1;5-D.

(a) Y1H assay showing that the four selected TFs are able to bind to the TaHKT1;5-D promoter. SD/−Leu, SD medium without Leu; SD/−Leu/AbA100, SD medium without Leu supplemented with AbA at the concentration of indicated 100 ng mL−1. Transformed yeast cells were dotted at 10−1 dilutions on the selective medium. Similar results were seen in three independent experiments. (b) Schematic diagram of motifs in the TaHKT1;5-D promoter region and DNA fragments (P1 and P2) for ChIP–qPCR. The black arrow indicates the transcription start site. (c) ChIP–qPCR showing in vivo binding of the TaHKT1;5-D promoter by TaNAC6 (using P2), TabHLH148 (using P1), TaGT2 (using P2) and TaMYB30 (using P2), respectively, in wheat root protoplasts. Each bar represents the mean ± SD (n = 3 independent experiments). (d,e) Dual-luciferase transient expression assay showing that TaNAC6, TabHLH148, TaGT2 and TaMYB30 can activate the promoter activity of TaHKT1;5-D. Scale bar in d, 1 cm. Each bar in e represents the mean ± SD (n = 8 biologically independent samples). EV, empty vector. In c and e, significance analysis was performed by two-tailed Student’s t test.

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Extended Data Fig. 9 Knockout of TtSPL6-A or TtSPL6-B in tetraploid wheat shows an adverse effect on yield-related traits.

(a) The structure and grain yield of spikes observed in the wild-type (WT), Ttspl6-aa and Ttspl6-bb lines. Scale bars, 1 cm. (b) Spikelets per spike, (c) kernels per spike and (d) grain yield per spike of WT, Ttspl6-aa and Ttspl6-bb lines. Each bar represents the mean ± SD (n = 8 biologically independent samples). In b–d, Significance analysis was performed by one-way Tukey multiple comparisons test.

Source data

Extended Data Fig. 10 Field performance of introgression lines harboring TaSPL6-DIn or TaSPL6-DDel on saline farmlands in Qingdao, China, and TaSPL6-DDel knockout lines in Dongying and Qingdao, China.

(a) The transcript abundance of TaHKT1;5-D in root-stelar cells and (b) whole-seedling biomass of sibling lines harboring TaSPL6-DIn or TaSPL6-DDel in hydroponic experiments. Each bar represents the mean ± SD (a, n = 3 independent experiments; b, n = 7 biologically independent samples). (c–f) Field performance of introgression lines harboring TaSPL6-DIn or TaSPL6-DDel on saline farmlands in Qingdao, China. (c) The flag leaf Na+ content, (d) kernels per spike, (e) thousand kernels weight and (f) grain yield per block of TaSPL6-DIn and TaSPL6-DDel lines. Each bar represents the mean ± SD (c, n = 9 biologically independent samples; d, n = 20 biologically independent samples; e, n = 8 biologically independent samples; f, n = 3 independent experiments). (g–j) Field performance of wild-type (WT) and two TaSPL6-DDel knockout lines on saline farmlands in Dongying, China. (g) The flag leaf Na+ content, (h) kernels per spike, (i) thousand kernels weight and (j) grain yield per block of WT and two TaSPL6-DDel knockout lines. Each bar represents the mean ± SD (g, n = 9 biologically independent samples; h, n = 20 biologically independent samples; i, n = 8 biologically independent samples; j, n = 3 independent experiments). (k–n) Field performance of wild-type (WT) and two TaSPL6-DDel knockout lines on saline farmlands in Qingdao, China. (k) The flag leaf Na+ content, (l) kernels per spike, (m) thousand kernels weight and (n) grain yield per block of WT and two TaSPL6-DDel knockout lines. Each bar represents the mean ± SD (k, n = 9 biologically independent samples; l, n = 20 biologically independent samples; m, n = 8 biologically independent samples; n, n = 3 independent experiments). In the box plots (d, h and l), centerline represents median and bounds of box indicate the interquartile range. In a–n, significance analysis was performed by two-tailed Student’s t test.

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Supplementary information

Supplementary Information

Supplementary Note, Supplementary Figs. 1–6 and supporting data for Supplementary Figs. 3 and 6.

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Statistical supporting data of Supplementary Figs. 4–6.

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Wang, M., Cheng, J., Wu, J. et al. Variation in TaSPL6-D confers salinity tolerance in bread wheat by activating TaHKT1;5-D while preserving yield-related traits. Nat Genet (2024). https://doi.org/10.1038/s41588-024-01762-2

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