Tomato QM-like protein protects Saccharomyces cerevisiae cells against oxidative stress by regulating intracellular proline levels

Abstract

Exogenous proline can protect cells of Saccharomyces cerevisiae from oxidative stress. We altered intracellular proline levels by overexpressing the proline dehydrogenase gene (PUT1) of S. cerevisiae. Put1p performs the first enzymatic step of proline degradation in S. cerevisiae. Overexpression of Put1p results in low proline levels and hypersensitivity to oxidants, such as hydrogen peroxide and paraquat. A put1-disrupted yeast mutant deficient in Put1p activity has increased protection from oxidative stress and increased proline levels. Following a conditional life/death screen in yeast, we identified a tomato (Lycopersicon esculentum) gene encoding a QM-like protein (tQM) and found that stable expression of tQM in the Put1p-overexpressing strain conferred protection against oxidative damage from H2O2, paraquat, and heat. This protection was correlated with reactive oxygen species (ROS) reduction and increased proline accumulation. A yeast two-hybrid system assay was used to show that tQM physically interacts with Put1p in yeast, suggesting that tQM is directly involved in modulating proline levels. tQM also can rescue yeast from the lethality mediated by the mammalian proapoptotic protein Bax, through the inhibition of ROS generation. Our results suggest that tQM is a component of various stress response pathways and may function in proline-mediated stress tolerance in plants.

FIG. 1.

Altered Put1p expression in yeast affects cell viability during oxidative stress. EGY48-pLexA vector, EGY48-pLexA-PUT1, and put1 deletion strains were spotted at fivefold serial dilutions on plates containing or not containing 1 mM paraquat. Growth was monitored after 3 days incubation at 30°C.

FIG. 2.

The tQM protein rescues yeast from oxidant-induced cell death. (A) The EGY48 yeast strain was transformed with an empty pB42AD vector (vector), pLexA-PUT1-expressing Put1p under control of a galactose-inducible promoter (PUT1), and constructs pLexA-PUT1 and pB42AD-tQM, which encodes tQM (PUT1 + tQM). Also shown is the expression of tQM alone in the EGY48 strain (tQM). Cultures of different transformants were diluted to equal densities. Fivefold serial dilutions of each culture were directly spotted onto SD-galactose medium supplemented with 1 mM H₂O₂. Pictures were taken after 3 days of growth. (B) Yeast strains as shown in panel A were inoculated on liquid SD medium containing 5 mM H₂O₂ and then grown for different time periods. Aliquots of each yeast strain at each time point were spread on the complete medium, and the number of viable colonies that grew after 3 days was counted. Results are expressed as the percent colonies surviving after H₂O₂ treatment relative to colonies without H₂O₂ treatment. Data shown are an average of three independent experiments with standard errors of <10%.

FIG. 3.

The tomato tQM gene product inhibits Bax-induced cell death in yeast. (A) The EGY48 strain was individually transformed with the empty vector pB42AD, pB42AD-tQM, pLexA-Bax, or pLexA-Bax plus pB42AD-tQM, and transformants were maintained on SD-galactose medium. A time course analysis of dead cell accumulation was employed during Bax-mediated yeast lethality. Evans blue staining was performed at 0, 4, 7, 12, and 24 h after culturing on SD-galactose medium. Dead cells were scored, and data shown are means ± standard errors of the mean of at least three independent experiments. (B) Transformants were treated with 50 μM DHR123 after 24 h of growth on SD-galactose medium and then observed with an epifluorescence microscope. The EGY48 strain was transformed with empty vector pB42AD (a), pB42AD-tQM (b), pLexA-Bax (c). or pLexA-Bax plus pB42AD-tQM (d).