Cloning and functional characterization of a vacuolar Na+/H+ antiporter gene from mungbean (VrNHX1) and its ectopic expression enhanced salt tolerance in Arabidopsis thaliana

Abstract

Plant vacuolar NHX exchangers play a significant role in adaption to salt stress by compartmentalizing excess cytosolic Na+ into vacuoles and maintaining cellular homeostasis and ionic equilibrium. We cloned an orthologue of the vacuolar Na+/H+ antiporter gene, VrNHX1 from mungbean (Vigna radiata), an important Asiatic grain legume. The VrNHX1 (Genbank Accession number JN656211.1) contains 2095 nucleotides with an open reading frame of 1629 nucleotides encoding a predicted protein of 542 amino acids with a deduced molecular mass of 59.6 kDa. The consensus amiloride binding motif (84LFFIYLLPPI93) was observed in the third putative transmembrane domain of VrNHX1. Bioinformatic and phylogenetic analysis clearly suggested that VrNHX1 had high similarity to those of orthologs belonging to Class-I clade of plant NHX exchangers in leguminous crops. VrNHX1 could be strongly induced by salt stress in mungbean as the expression in roots significantly increased in presence of 200 mM NaCl with concomitant accumulation of total [Na+]. Induction of VrNHX1 was also observed under cold and dehydration stress, indicating a possible cross talk between various abiotic stresses. Heterologous expression in salt sensitive yeast mutant AXT3 complemented for the loss of yeast vacuolar NHX1 under NaCl, KCl and LiCl stress indicating that VrNHX1 was the orthologue of ScNHX1. Further, AXT3 cells expressing VrNHX1 survived under low pH environment and displayed vacuolar alkalinization analyzed using pH sensitive fluorescent dye BCECF-AM. The constitutive and stress inducible expression of VrNHX1 resulted in enhanced salt tolerance in transgenic Arabidopsis thaliana lines. Our work suggested that VrNHX1 was a salt tolerance determinant in mungbean.

Figure 1. The phylogenetic tree for plant Na⁺/H⁺ antiporters was generated using MEGA4: Tree Explorer software.

The evolutionary history was inferred using the neighbor-joining method and analyzed using bootstrap analysis with 500 replicates. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and are in the units of the number of amino acid substitutions per site. The GenBank Accession numbers for NHX proteins used are: VrNHX1 (AEO50758.1), VuNHX1 (AEO72079.2), GmNHX1 (AAY430061.1), CkNHX1 (ABG89337.1), MsNHX1 (AAS84487.1), CaNHX1 (ADL28385.1), TrNHX1 (ABV00895.1), LtNHX1 (ACE78322.1), AhNHX1 (ADK74832.1), AtNHX1 (NM_122597.2).

Figure 2. Copy number analysis of VrNHX1 in mungbean genome.

Mungbean genomic DNA (20 µg) was digested with EcoRI and HindIII, and hybridized with DIG-labeled probe corresponding to the CDS of VrNHX1. Hybridization signals are indicated as arrows.

Figure 3. Cation sensitivity assay of transformed yeast strains (W303-1B, AXTYES2.0, AXTVrNHX1) under various concentrations of NaCl, KCl, and LiCl.

Saturated seed cultures for each strain was diluted to an OD₆₀₀ of 0.006 and inoculated to liquid APGal medium (pH 5.5) supplemented with or without various concentrations of (A) NaCl (0, 50, 75, 100 mM), B) KCl (0, 0.5, 0.75, 1.0 M), and (C) LiCl (0, 15, 20, 25 mM). Growth was observed at 30°C after 3 days and absorbance recorded at 600 nm. Data are means of 3 independent events (n = 3) and standard errors are plotted in the graph. Statistically significant values at P≤0.05 are indicated as “*”, using Bonferroni analysis.

Figure 4. Heterologous expression of VrNHX1 in yeast mutant.

Wild type (W303-1B) strain was used as a control, Δ ena1- 4 Δnha1 Δnhx1 mutant (AXT3) strain was transformed with null pYES2.0 (labeled as AXTYES2.0 strain) and pYESVrNHX1 recombinant vector (labeled as AXTVrNHX1) were used for complementation assay. 10-fold serial dilutions of saturated seed cultures of each strain were spotted onto APGal media (pH-5.5) supplemented with or without (A) 50, 75 and 100 mM NaCl, (B) 25 mM LiCl, and (B) 0.5 M KCl. (C) Hygromycin sensitivity assay was performed by spotting 10-fold serial dilutions of saturated seed cultures of each strain onto YPGal media (pH- 5.5) supplemented with or without 50 µg/ml Hyg. The plates were incubated at 30°C for 3 days.

Figure 5. Total intracellular ion estimation in yeast strains W303-1B, AXTYES2.0 and AXTVrNHX1.

Yeast cells were grown in APG medium (pH 4.0) with 1 mM KCl supplemented in presence (stressed) or absence of 75 mM NaCl (unstressed) and harvested at a cell density of 0.3. Total intracellular, vacuolar and cytoplasmic Na⁺ and K⁺ content was determined as described in the materials and methods section. Data are means of 3 independent events (n = 3) and standard errors are plotted in the graph. Statistically significant values at P≤0.05 are indicated as “*”, using Bonferroni analysis.

Figure 6. Measurement of vacuolar pH in yeast strains.

(A) Vacuolar pH was measured for BCECF-AM loaded yeast strains W303-1B, AXTYES2.0 and AXTVrNHX1 as described in materials and methods following the calibration curve (Figure S4). Mean and SEs are plotted for three independent events (n = 3) in each case. Statistically significant values at P≤0.05 are indicated as “*”, using Bonferroni analysis. (B) Accumulation of pH-sensitive fluorescent BCECF dye in yeast vacuoles was measured. The yeast strains were grown in APGal media (pH 5.0), resuspended in minimal medium with BCECF-AM dye for 30 min at 30°C. Yeast cells were visualized by Nikon eclipse Ti-U Fluorescence microscope (Nikon) at excitation wavelength of 440 nm. Bar scale: 50 µm.

Figure 7. Expression analysis of VrNHX1 in early and mid stage mungbean seedlings under various abiotic stresses.

(A) Semi-quantitative RT-PCR for studying expression patterns of VrNHX1 under salt stress was performed. Total RNA was isolated from leaves and roots of early (5 days) and mid stage mungbean seedlings (10 days) under 200 mM NaCl treatment at time intervals of 0, 6, 12, 18, 24, and 48 hrs. (B) Semi-quantitative RT-PCR for studying expression patterns of VrNHX1 under different abiotic stress conditions such as salt, cold and dehydration stress was studied. Total RNA was isolated from mid stage mungbean seedlings under (A) 200 mM NaCl, (B) Cold (4°C), and (C) 200 mM Mannitol treatment at time intervals of 0, 6, 12, and 24 hrs. PCR fragments of 566 bp and 422 bp size corresponding to VrNHX1 and VrTubβ were fractionated electrophoretically on 2% agarose gel stained with 10 mg/ml ethidium bromide.

Figure 8. Total intracellular ion measurement in leaves and roots of early and mid stage mungbean seedlings.

Na⁺ and K⁺ content in (A) leaves and (B) roots of unstressed and salt stressed mungbean seedlings harvested at time intervals of 0, 6, 12, 24, 48, and 72 hrs was measured using Flame Photometer. Values indicate means ± SE (n = 3). Statistically significant values at P≤0.05 are indicated as “*”, using Bonferroni analysis.

Figure 9. Effect of salt stress on germination efficiency and root growth of transgenic Arabidopsis lines.

(A) The wildtype (WT, col-0) and transgenic (line 1, 35S::VrNHX1 and line 4, RD29A::VrNHX1) seedlings were observed for germination score after 10 days exposure to salt stress (150 mM NaCl). (B) Root growth inhibition in wild type (WT, Col-0) and transgenic Arabidopsis (Line 1, 35S::VrNHX1 and Line 4, RD29A::VrNHX1) plants upon salt stress (150 mM NaCl) was studied. The 4 days old germinated seedlings were transferred to 150 mM NaCl stress for a period of 7 days and (C) root length measured was plotted in graph. Values indicate means ± SE (n = 10). Statistically significant values at P≤0.05 are indicated as “*”, using Bonferroni analysis.

Figure 10. Studying the physiological changes in transgenic Arabidopsis lines under salt stress.

(A) Effect of salt stress in wild type (WT, Col-0) and transgenic Arabidopsis lines expressing VrNHX1 constitutively (Lines 1–3, 35S::VrNHX1) and inducibly (Lines 4–6, RD29A::VrNHX1). NaCl-induced morphological changes was visible in 10 days old WT and transgenic lines after exposure to 200 mM NaCl for 5 days. (B) Changes in chlorophyll, MDA and proline content were estimated and analyzed as explained in materials and methods section. Values indicate means ± SE (n = 3). Statistically significant values at P≤0.05 are indicated as “*”, using Bonferroni analysis.

Figure 11. Salt tolerance assay in mature transgenic Arabidopsis lines under salt stress.

(A) Effect of salt stress on wild type (WT, Col-0) and transgenic Arabidopsis lines expressing VrNHX1 constitutively (Line 1, 35S::VrNHX1) and inducibly (Line 4, RD29A::VrNHX1) subjected to 250 mM NaCl treatment for 2 weeks (B) Relative transgene expression level of VrNHX1 in transgenic Arabidopsis lines under unstressed and salt stressed conditions. No transgene expression was observed in WT. A 0.283 kb fragment of VrNHX1::35SployA and 0.150 kb fragment of AtUBQ1 was amplified in quantitative RT-PCR analysis (C) Na⁺ and (D) K⁺ content (µmoles/g DW) was estimated in leaves of unstressed (0 mM NaCl) and salt stressed (250 mM NaCl) WT and transgenic lines, as described in materials and methods. Values indicate means ± SE (n = 3). Statistically significant values at P≤0.05 are indicated as “*”, using Bonferroni analysis.