Overexpression of a Barley Aquaporin Gene, HvPIP2;5 Confers Salt and Osmotic Stress Tolerance in Yeast and Plants

Abstract

We characterized an aquaporin gene HvPIP2;5 from Hordeum vulgare and investigated its physiological roles in heterologous expression systems, yeast and Arabidopsis, under high salt and high osmotic stress conditions. In yeast, the expression of HvPIP2;5 enhanced abiotic stress tolerance under high salt and high osmotic conditions. Arabidopsis plants overexpressing HvPIP2;5 also showed better stress tolerance in germination and root growth under high salt and high osmotic stresses than the wild type (WT). HvPIP2;5 overexpressing plants were able to survive and recover after a 3-week drought period unlike the control plants which wilted and died during stress treatment. Indeed, overexpression of HvPIP2;5 caused higher retention of chlorophylls and water under salt and osmotic stresses than did control. We also observed lower accumulation of reactive oxygen species (ROS) and malondialdehyde (MDA), an end-product of lipid peroxidation in HvPIP2;5 overexpressing plants than in WT. These results suggest that HvPIP2;5 overexpression brought about stress tolerance, at least in part, by reducing the secondary oxidative stress caused by salt and osmotic stresses. Consistent with these stress tolerant phenotypes, HvPIP2;5 overexpressing Arabidopsis lines showed higher expression and activities of ROS scavenging enzymes such as catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR), and ascorbate peroxidase (APX) under salt and osmotic stresses than did WT. In addition, the proline biosynthesis genes, Δ ¹-Pyrroline-5-Carboxylate Synthase 1 and 2 (P5CS1 and P5CS2) were up-regulated in HvPIP2;5 overexpressing plants under salt and osmotic stresses, which coincided with increased levels of the osmoprotectant proline. Together, these results suggested that HvPIP2;5 overexpression enhanced stress tolerance to high salt and high osmotic stresses by increasing activities and/or expression of ROS scavenging enzymes and osmoprotectant biosynthetic genes.

Keywords: Arabidopsis; HvPIP2;5; aquaporin; barley; overexpression; stress tolerance; yeast.

Figure 1

Effect of HvPIP2;5 expression in yeast under salt and osmotic stresses. Yeast cells harboring the HvPIP2;5 expressing construct (HvPIP2;5), yeast cells only (yeast), and yeast cells with the vector pYES2 only (vector only) were subjected to 200 mM NaCl and 4% PEG. Cell density was adjusted to OD-600 at 1.0 and serial dilutions were made at each step. Ten microliter of each dilution was spotted on (A) YPD plates without stress. (B) YPD plates supplemented with 200 mM NaCl. (C) Supplemented with 4% PEG. Photographs were taken after 48 h of incubation at 30°C.

Figure 2

Phenotypes of HvPIP2;5 overexpressing Arabidopsis under salt and water-deficit stresses. WT and two HvPIP2;5 overexpressing Arabidopsis lines (OE1,OE2) were used for analysis. (A,D) Seed germination of WT and OE lines on MS media containing 200 mM NaCl or 20% PEG. The number of germinated seeds was expressed as a percentage of total number of seeds planted. (B,E) Root elongation on WT and OE lines on MS media containing 200 mM NaCl or 20% PEG. Pictures for root elongation comparison were taken 21 days after planting. (C) Effects of drought on WT and OE lines. Four week old seedlings were subjected to drought stress by withholding water supply for 21 days and then re-watered. (F) Measurement of chlorophyll A in WT and OE lines after 200 mM NaCl or 20% PEG treatment. Bars indicate standard error and significant differences between WT and OE lines were marked with asterisks (P < 0.05).

Figure 3

Water loss and retention in HvPIP2;5 overexpressing Arabidopsis under water stress. (A) Comparison of water loss from detached rosette leaves of WT and OE lines, control plants. Water loss was calculated from the loss in fresh weight of the samples. (B) Relative water content estimation in roots of WT and OE lines after 200 mM NaCl or 20% PEG treatment. Bars indicate standard error and significant differences between WT and OE lines were marked with asterisks (P < 0.05).

Figure 4

Analysis of superoxide, hydrogen peroxide, malondialdehyde contents in HvPIP2;5 overexpressing lines under salt and osmotic stresses. (A) NBT (nitro blue tetrazolium) staining for superoxide detection in WT and OE lines after NaCl treatment. (B) Quantification of superoxide contents in WT and OE lines after 200 mM NaCl or 20% PEG treatment. (C) DAB (3, 3-diaminobenzidine) staining for hydrogen peroxide detection in WT and OE lines after NaCl treatment. (D) Quantification of hydrogen peroxide contents in WT and OE lines after 200 mM NaCl or 20% PEG treatment. (E) Quantification of MDA (malondialdehyde) levels in WT and OE lines after 200 mM NaCl or 20% PEG treatment. Bars indicate standard error and significant differences between WT and OE lines were marked with asterisks (P < 0.05).

Figure 5

Activities of reactive oxygen species scavenging enzymes in HvPIP2;5 overexpressing lines under salt and osmotic stresses. (A) Activities of catalase (CAT) in WT and OE lines after 200 mM NaCl or 20% PEG treatment. (B) Activities of superoxide dismutase (SOD) in WT and OE lines after 200 mM NaCl or 20% PEG treatment. (C) Activities of glutathione reductase after NaCl treatments in WT and OE lines. (D) Activities of ascorbate peroxidase after NaCl treatments in WT and OE lines. Bars indicate standard error and significant differences between WT and OE lines were marked with asterisks (P < 0.05).

Figure 6

Analysis of P5CS1 and P5CS2 gene expression and proline levels in HvPIP2;5 overexpressing lines under salt and osmotic stresses. The expression levels of (A,B) P5CS1 and P5CS2 in WT and OE lines were estimated by quantitative real time PCR. Arabidopsis clatharin gene was used as an internal control for normalization. The expression levels of each gene in WT control (calibrator) were assumed as 1. Three biological replicates were averaged and bars indicate standard error. (C) Proline contents in WT and OE were quantified with three biological replicates. Bars indicate standard error and significant differences between WT and OE lines were marked with asterisks (P < 0.05).