The Thellungiella salsuginea tonoplast aquaporin TsTIP1;2 functions in protection against multiple abiotic stresses

Abstract

Examination of aquaporin (AQP) membrane channels in extremophile plants may increase our understanding of plant tolerance to high salt, drought or other conditions. Here, we cloned a tonoplast AQP gene (TsTIP1;2) from the halophyte Thellungiella salsuginea and characterized its biological functions. TsTIP1;2 transcripts accumulate to high levels in several organs, increasing in response to multiple external stimuli. Ectopic overexpression of TsTIP1;2 in Arabidopsis significantly increased plant tolerance to drought, salt and oxidative stresses. TsTIP1;2 had water channel activity when expressed in Xenopus oocytes. TsTIP1;2 was also able to conduct H₂O₂ molecules into yeast cells in response to oxidative stress. TsTIP1;2 was not permeable to Na(+) in Xenopus oocytes, but it could facilitate the entry of Na(+) ions into plant cell vacuoles by an indirect process under high-salinity conditions. Collectively, these data showed that TsTIP1;2 could mediate the conduction of both H₂O and H₂O₂ across membranes, and may act as a multifunctional contributor to survival of T. salsuginea in highly stressful habitats.

Keywords: Aquaporin; Channeling activity; Stress tolerance; Thellungiella salsuginea.

Fig. 1

Subcellular localization of TsTIP1;2–GFP fusion proteins in BY2 cells. (A) GFP fluorescence in control BY2 cells. (B) GFP fluorescence in TsTIP1;2-GFP transgenic BY2 cells. (C) Protoplast derived from a TsTIP1;2-GFP transgenic BY2 cell. (D) Vacuole released from the protoplast derived from the TsTIP1;2-GFP transgenic BY2 cell after short mechanical pressure. The GFP was visualized under a laser scanning confocal microscope at an excitation wavelength of 488 nm. Scale bars = 10 µm.

Fig. 2

Expression patterns of TsTIP1;2. (A) Expression of TsTIP1;2 in various organs of T. salsuginea. (B–F) Time course analysis of TsTIP1;2 expression in response to abiotic stresses. Total RNA was isolated from 2-week-old plants treated with drought stress (B), 300 mM NaCl (C), 25 µM MV (D), 10 mM H₂O₂ (E) or 50 µM ABA (F), for 0, 1, 3, 6, 12 and 24 h, respectively. The levels of TsTIP1;2 and TsActin2 were measured by SYBR Green I real-time PCR. The 0 h value was used as reference. Each column represents means ± SE from three biological replicates.

Fig. 3

Effects of drought stress on wild-type (WT) and TsTIP1;2 transgenic Arabidopsis plants. (A) Expression of TsTIP1;2 in WT and transgenic plants (lines T14, T19 and T15). (B) Phenotypic comparison of the WT and transgenic plants before and after drought stress treatment. Upper panel: plants grown under normal conditions. Lower panel: plants subjected to water withholding for 4 weeks. (C) Survival rate of plants under drought stress. Each value is the average of three biological experiments (n = 30 for each experiment) and bars indicate the SE. (D) Malondialdehyde (MDA) contents in Arabidopsis plants growing under normal conditions or after drought treatment for 1 week. Each data point represents the mean ± SE. Three biological repeats were performed. Asterisks indicate signiflcant differences compared with wild-type plants (*P < 0.05, **P < 0.01, Student’s t-test). (E) Relative water contents (RWCs) in aerial parts of the pot-grown plants before or after withholding water for 1 week. (F) Water loss rate in aerial parts of the pot-grown plants. For E and F, error bars indicate the SE and each value is the average of four experiments. Asterisks indicate signiflcant differences compared with the WT control (*P < 0.05, Student’s t-test).

Fig. 4

Effects of salt stress on wild-type (WT) and TsTIP1;2 transgenic Arabidopsis plants. (A) Phenotype of WT and TsTIP1;2 transgenic plants (lines T15, T14 and T19) recovered from salt stress treatment. Five-day-old WT and transgenic seedlings were transferred to 1/2 MS solid medium supplemented with 150 mM NaCl and grown for 1 week and then recovered on 1/2 MS solid medium for 10 d. (B) Survival rate of the salt-treated plants. (C) Survival rate of the salt-treated plants after recovery. For B and C, each value is the average of three biological experiments (n = 30 for each experiment) and bars indicate the SE. (D) MDA levels in plants. Each data point represents the mean ± SE from three biological repeats. Asterisks indicate a significant difference compared with WT plants (*P < 0.05, **P < 0.01, Student’s t-test).

Fig. 5

Effects of oxidative stress on wild-type (WT) and TsTIP1;2 transgenic Arabidopsis plants. (A) Phenotype of WT and TsTIP1;2 transgenic plants (lines T15, T14 and T19) recovered from oxidative stress treatment. Five-day-old WT and transgenic seedlings were transferred to 1/2 MS solid medium supplemented with 1 µM MV and grown for 1 week and then recovered on 1/2 MS solid medium for 10 d. (B) MDA contents in plants. Each data point represents the mean ± SE from three biological repeats. (C) Relative fresh weight of plants under MV stress, as a percentage of weight of unstressed plants. (D) Relative Chl contents in plants under MV stress, as a percentage of Chl content in unstressed plants. For C and D, each data point is the average of three experiments (n = 15 for each experiment) and bars indicate the SE (*P < 0.05, **P < 0.01, Student’s t-test).

Fig. 6

Water channel activity assessment of TsTIP1;2. (A) The swelling kinetics of Xenopus oocytes injected with H₂O, or cRNA coding for AtTIP1;1 and TsTIP1;2, respectively. The water-injected oocytes were used as a negative control. AtTIP1;1-injected oocytes were used as a positive control. V/V₀ represents the volume changes upon immersion in hypo-osmotic solution. V₀ represents the volume at the initial time. Three biological replicates were performed. Bars indicate the SE. (B) Water permeability coefficient (Pf) of H₂O, TsTIP1;2 and AtTIP1;1. The Pf values were calculated according to the equation described by Zhang and Verkman. (1991). Asterisks indicate significant differences in comparison with oocytes injected with water (*P < 0.05, **P < 0.01, Student’s t-test).

Fig. 7

H₂O₂ permeability of yeast cells expressing TsTIP1;2. (A) Saccharomyces cerevisiae aqy-null strain cells transformed with the empty vector pYES2 alone or pYES2-TsTIP1;2 were spotted in 10-fold dilutions on medium without or with 1, 1.5 or 2 mM H₂O₂, respectively. The S. cerevisiae aqy-null strain transformed with the empty vector pYES2 was used as the control. (B) TsTIP1;2-mediated H₂O₂ diffusion across yeast membranes. The fluorescence of CM-H₂DCFDA-loaded S. cerevisiae aqy-null cells transformed with TsTIP1;2 or empty vector pYES2 was recorded over time. The assay was performed for 30 min after addition of 10 mM H₂O₂. (C) Bar diagram showing the average increase in fluorescence. Cells were treated with 2 or 10 mM H₂O₂ for 30 min before fluorescence measurement. White bars, control; black bars, treatment with 2 mM H₂O₂; dark gray bars, treatment with 10 mM H₂O₂; light gray bars, pre-incubation with 10 µM AgNO₃ prior to 10 mM H₂O₂ treatment. The experiment was repeated more than three times.

Fig. 8

Assessment of Na⁺content in wild-type (WT) and TsTIP1;2 transgenic plants (lines T15, T14 and T19). (A) Na⁺ content in plant cells was measured using the Na⁺ fluorescent dye indicator Sodium Green before and after treatment with 300 mM NaCl. Scale bar = 50 µm. (B) Quantitative analysis of Na⁺ levels in plants. Error bars indicate the SE of more than four biological repeats. Asterisks indicate a significant difference in comparison with the wild-type plants (*P < 0.05, **P < 0.01, Student’s t-test).