Overexpression of a Metallothionein 2A Gene from Date Palm Confers Abiotic Stress Tolerance to Yeast and Arabidopsis thaliana

Abstract

Although the date palm tree is an extremophile with tolerance to drought and certain levels of salinity, the damage caused by extreme salt concentrations in the soil, has created a need to explore stress-responsive traits and decode their mechanisms. Metallothioneins (MTs) are low-molecular-weight cysteine-rich proteins that are known to play a role in decreasing oxidative damage during abiotic stress conditions. Our previous study identified date palm metallothionein 2A (PdMT2A) as a salt-responsive gene, which has been functionally characterized in yeast and Arabidopsis in this study. The recombinant PdMT2A protein produced in Escherichia coli showed high reactivity against the substrate 5'-dithiobis-2-nitrobenzoic acid (DTNB), implying that the protein has the property of scavenging reactive oxygen species (ROS). Heterologous overexpression of PdMT2A in yeast (Saccharomyces cerevisiae) conferred tolerance to drought, salinity and oxidative stresses. The PdMT2A gene was also overexpressed in Arabidopsis, to assess its stress protective function in planta. Compared to the wild-type control, the transgenic plants accumulated less Na⁺ and maintained a high K⁺/Na⁺ ratio, which could be attributed to the regulatory role of the transgene on transporters such as HKT, as demonstrated by qPCR assay. In addition, transgenic lines exhibited higher chlorophyll content, higher superoxide dismutase (SOD) activity and improved scavenging ability for reactive oxygen species (ROS), coupled with a better survival rate during salt stress conditions. Similarly, the transgenic plants also displayed better drought and oxidative stress tolerance. Collectively, both in vitro and in planta studies revealed a role for PdMT2A in salt, drought, and oxidative stress tolerance.

Keywords: abiotic stress; date palm; drought; functional characterization; metallothionein; salinity.

Figure 1

Sequence analysis of 10 metallothioneins (MT) amino acid sequences of various plant species and the phylogenetic tree constructed using the neighbor-joining method. The bootstrap values on the nodes represent percentages of 1000 repetitions (A). The multiple sequence alignment of the deduced amino acid sequence of PdMT2A and other MT2A isoforms from other plant species. The gray-colored highlighting represents identical and conserved regions and the dark highlighting represents similar regions with one amino acid difference. The two cysteine-rich domains are indicated at the N- and C-termini of the sequences (B). The hydrophobicity plot of each amino acid in the PdMT2A sequence according to the Kyte–Doolittle hydrophobicity scale (C). The sequence analysis of the putative 2000-bp promoter region upstream of the PdMT2A start codon, showing the abundance of abiotic-stress-related transcription factor binding sites (TFBSs) within the putative promoter sequence (D).

Figure 2

Production of recombinant PdMT2A protein in E. coli. Schematic representation of the expression vector (A). The polyacrylamide gel image of the partially purified recombinant PdMT2A protein and the protein produced by the pTYB21 empty vector in E. coli. (B). The reactivity of the recombinant PdMT2A protein and the empty vector against the 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) substrate shows the initial speed of the reaction from the plot of absorbance versus time (C).

Figure 3

Overexpression of PdMT2A in yeast. Schematic representation of the cloned PdMT2A gene within the pYES-DEST52 plasmid (A). The effect of the PdMT2A transgene on the growth of yeast cells and the relative tolerance of transgenic (TY) and empty vector (EV) cells tested by yeast spot assay when grown under control and various abiotic stress conditions on solid media (B). Yeast liquid culture assay used to test the relative tolerance of TY and EV cells grown under control and 50 mM NaCl stress conditions (C). Accumulation of Na⁺ and K⁺ in TY or EV cells when grown under control and 25 mM NaCl salinity stress conditions (D). The bars represent the mean concentration of Na⁺ and K⁺ (± SE, n = 3).

Figure 4

Overexpression of PdMT2A in Arabidopsis. Schematic representation of the cloned PdMT2A gene within the pEarleyGate 203 plasmid (A). Dot-blot immunoassay using total protein lysate extracted from the transgenic and the wild-type (WT) Arabidopsis plants, to demonstrate the heterologous expression of Myc-PdMT2A protein and the actin protein, using anti-Myc and anti-actin antibodies, respectively (B).

Figure 5

The effect of PdMT2A on the phenotype of the transgenic Arabidopsis seedlings. The growth pattern, root length and dry weight of the WT and PdMT2A transgenic Arabidopsis lines were measured when the seedlings were grown on plain half-MS plates as a control (A) or on half-MS plates supplied with 100 mM NaCl (B), 150 mM mannitol (C) or 2 mM H₂O₂ (D), for 14 days. The length of the main root was measured manually using a standard centimeter scale. The bars represent either the mean root length in cm or the dry weight in mg (± SE, n = 3), while the asterisks indicate a significant difference from WT plants (p < 0.05).

Figure 6

The effect of PdMT2A on the accumulation of Na⁺ and K⁺ content in Arabidopsis. Accumulation of Na⁺ (A) and K⁺ (B) and the K⁺/Na⁺ ratio (C) in PdMT2A transgenic and WT Arabidopsis plants when subjected to control, drought (150 mM mannitol) and salinity (100 mM NaCl) conditions on half-strength MS-medium plates. The bars represent the mean concentrations of Na⁺ and K⁺ (± SE, n = 3), while the asterisks indicate a significant difference from the WT plants (p < 0.05).

Figure 7

The effect of PdMT2A on chlorophyll, proline and antioxidant enzymes. The effect of drought, salinity and oxidative stress on the chlorophyll content (A), proline concentration (B), malondialdehyde (MDA) concentration (C), superoxide dismutase (SOD) activity (D) and ascorbate peroxidase (APX) activity (E) of PdMT2A transgenic and WT Arabidopsis plants. The bars represent the mean chlorophyll, proline or MDA concentration and the SOD or APX activity (± SE, n = 3), while the asterisks indicate a significant difference from the WT plants (p < 0.05).

Figure 8

Performance of the PdMT2A transgenic and WT Arabidopsis plants grown on soil and subjected to salinity (200 mM NaCl) and drought stress for 14 days.

Figure 9

The 3,3-diaminobenzidine (DAB) and nitro blue tetrazolium (NBT) histochemical staining of WT and transgenic Arabidopsis plant leaves under salinity and drought stress conditions.

Figure 10

Gene expression analysis of CHX20, SOS1, HKT1, vacuolar Na⁺/H⁺ antiporter, ABA stress-induced gene and SOD in WT and transgenic Arabidopsis subjected to salinity and drought stress. The bars represent the relative fold change (± SE, n = 3), while the asterisks indicate a significant difference from the control (p < 0.05).