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A novel tonoplast Na+/H+ antiporter gene from date palm (PdNHX6) confers enhanced salt tolerance response in Arabidopsis

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A sodium hydrogen exchanger (NHX) gene from the date palm enhances tolerance to salinity in Arabidopsis plants.

Abstract

Plant sodium hydrogen exchangers/antiporters (NHXs) are pivotal regulators of intracellular Na+/K+ and pH homeostasis, which is essential for salt stress adaptation. In this study, a novel orthologue of Na+/H+ antiporter was isolated from date palm (PdNHX6) and functionally characterized in mutant yeast cells and Arabidopsis plants to assess the behavior of the transgenic organisms in response to salinity. Genetically transformed yeast cells with PdNHX6 were sensitive to salt stress when compared to the empty vector (EV) yeast cells. Besides, the acidity value of the vacuoles of the transformant yeast cells has significantly (p ≤ 0.05) increased, as indicated by the calibrated fluorescence intensity measurements and the fluorescence imagining analyses. This observation supports the notion that PdNHX6 might regulate proton pumping into the vacuole, a crucial salt tolerance mechanism in the plants. Consistently, the transient overexpression and subcellular localization revealed the accumulation of PdNHX6 in the tonoplast surrounding the central vacuole of Nicotiana benthamiana leaf epidermal cells. Stable overexpression of PdNHX6 in Arabidopsis plants enhanced tolerance to salt stress and retained significantly higher chlorophyll, water contents, and increased seed germination under salinity when compared to the wild-type plants. Despite the significant increase of Na+, transgenic Arabidopsis lines maintained a balanced Na+/K+ ratio under salt stress conditions. Together, the results obtained from this study imply that PdNHX6 is involved in the salt tolerance mechanism in plants by controlling K+ and pH homeostasis of the vacuoles.

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Acknowledgements

The authors would like to thank Professor Hana Sychrova, Institute of Physiology Academy of Sciences of the Czech Republic, Prague, Czech Republic, for donating the salt-sensitive mutant S. cerevisiae BYT458 strain, which was used in this study.

Funding

This study is supported by the generous grant number RC/RG-SCI/BIOL/18/01 from the research council (TRC), Oman to MWY.

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Authors and Affiliations

  1. Department of Biology, College of Sciences, Sultan Qaboos University, P.O. Box 36, 123, Muscat, Oman

    Ibtisam Al-Harrasi, Gerry Aplang Jana, Himanshu V. Patankar & Mahmoud W. Yaish

  2. Department of Crop Sciences, College of Agricultural and Marine Sciences, Sultan Qaboos University, P.O. Box 34, 123, Muscat, Oman

    Rashid Al-Yahyai

  3. Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore, 117543, Singapore

    Sivamathini Rajappa & Prakash P. Kumar

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  1. Ibtisam Al-Harrasi
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Correspondence to Mahmoud W. Yaish.

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Supplementary file1 (XLSX 9 kb)

299_2020_2549_MOESM2_ESM.tif

Supplementary file2 Multiple sequence alignment of the deduced amino acid sequence of date palm (PdNHX6) and seven NHX6 isoforms from different plant species. PdNHX6 protein conserved regions (dark grey-colored highlights) that are shared with other NHX6 members. All the shaded areas expand the common conserved domain of NHX family known as sodium/hydrogen exchanger 3 (TIF 5439 kb)

299_2020_2549_MOESM3_ESM.tif

Supplementary file3 The putative topology of date palm (PdNHX6) and Arabidopsis (AtNHX6), using Protter database. The toplogy of PdNHX6 shows 11 transmembrane domains and signal peptide (a). However, AtNHX6 putative toplogy shows 12 transmembrane domains and no signal peptide was predicted (b) (TIF 2393 kb)

299_2020_2549_MOESM4_ESM.tif

Supplementary file4 The abundance of transcription factor binding sites (TFBSs) within 2000 bp promoter region of PdNHX6 computationally analyzed using PlantPAN 2.0 database (TIF 711 kb)

299_2020_2549_MOESM5_ESM.tif

Supplementary file5 The calibration curve of normalized fluorescent intensity (NI490) plotted against pH ranging from 4.0–8.0 (TIF 582 kb)

299_2020_2549_MOESM6_ESM.tif

Supplementary file6 PCR-based genotyping of PdNHX6 transgenic Arabidopsis plants using different primer pairs; 35S promoter and NHX6RA (a), NHX6FA, and OCS terminator (b) NHX6FA and NHX6RA (c) and 35S promoter and OCS terminator (d) (TIF 996 kb)

299_2020_2549_MOESM7_ESM.tif

Supplementary file7 Expression analysis of three independent homozygous PdNHX6 transgenic Arabidopsis lines (TL1, TL2, and TL3) and the WT using semi-quantitative RT-PCR and shown on an ethidium bromide-stained 1% agarose gel. The Arabidopsis actin 2 (AtACT2) (AT3G18780) was used as a loading control (TIF 3180 kb)

299_2020_2549_MOESM8_ESM.tif

Supplementary file8 Assay of the salinity tolerance ability of PdNHX6 Arabidopsis lines in vitro. The effect of PdNHX6 on root length, fresh weight, and dry weight of Arabidopsis plantlets grown on MS-agar plates (a) and MS-agar plates supplemented with 100 mM NaCl (b). Each genotype is denoted by color code, as shown on top of the scanning images. The bars represent the mean root length ± SE of three independent replicates. The bars with different letters are significantly different at p < 0.05 (TIF 1588 kb)

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Al-Harrasi, I., Jana, G.A., Patankar, H.V. et al. A novel tonoplast Na+/H+ antiporter gene from date palm (PdNHX6) confers enhanced salt tolerance response in Arabidopsis. Plant Cell Rep 39, 1079–1093 (2020). https://doi.org/10.1007/s00299-020-02549-5

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