Reactive oxygen species (ROS) are produced by all aerobically respiring cells. ROS can have detrimental effects on cells by oxidizing lipids, proteins, DNA, and carbohydrates, resulting in disease and death (22
). It is therefore essential for aerobic organisms to modulate ROS levels and activities in order to protect against toxicity. The α-imino acid proline functions as a potent antioxidant by scavenging intracellular ROS generated by the phytopathogenic fungus Colletotrichum trifolii
). The protective role of proline could be extended to the budding yeast Saccharomyces cerevisiae
, since proline conferred cell survival in the presence of lethal levels of paraquat, a contact herbicide that uncouples electron transport by generating lethal levels of superoxide (7
). Proline also is a well-known osmoprotectant, capable of mitigating the impacts of drought, salt, and temperature stress in higher plants (11
). In S. cerevisiae
, the cryoprotective activity of proline was established through a positive correlation between intracellular proline levels and resistance to freeze stress (33
). These abiotic stresses, including drought, salinity, and cold, are tightly linked to ROS generation (2
). Thus, these findings suggest a positive correlation between intracellular proline levels and resistance to oxidative stress. However, the mechanisms of proline-mediated stress protection and, in particular, the components involved in proline-dependent signal transduction pathways are still not well understood.
Intracellular proline levels are controlled by a series of key proline metabolic enzymes mediating proline synthesis and degradation. In S. cerevisiae
, two enzymes, proline dehydrogenase (Put1p) and Δ1
-pyrroline-5-carboxylate (P5C) dehydrogenase (Put2p), mediate the conversion of proline to glutamate in the mitochondria (5
). Accumulating evidence has shown that these two enzymes also are active in proline-mediated stress responses. Proline accumulation by mutation or disruption of PUT1
enhances freeze tolerance and desiccation stresses (45
). Increased intracellular proline levels in a put1
mutant yeast strain also were correlated with higher tolerance to hydrogen peroxide (H2
). Thus, proline acts as a compatible solute and protects cells against damage during oxidative stress. Accumulation of the proline catabolic intermediate P5C by disruption of the PUT2
gene triggers intracellular ROS generation, which suggests that proline catabolism contributes to intracellular oxidative stress (36
The role of proline metabolic enzymes in oxidative stress also has been described in other organisms. In a human colon cancer cell line, proline dehydrogenase activity was induced by p53-dependent initiation of apoptosis and catalyzed proline-mediated ROS formation (12
). The antioxidant enzyme Mn-superoxide dismutase effectively inhibits apoptosis induced by proline dehydrogenase activity (27
). In Arabidopsis thaliana
, incompatible interactions with Pseudomonas syringae
pv. tomato, which generates high amounts of ROS, resulted in proline accumulation and transcriptional activation of AtP5CS, an enzyme involved in proline biosynthesis (15
). However, it remains unclear how these proline metabolic enzymes are regulated in response to oxidative stress.
The small, basic QM protein was first identified as a putative tumor suppressor from the Wilms' tumor cell line (13
). It is highly conserved in mammals, plants, worms, insects, and yeasts (16
). Recent studies of mammalian cells suggest that QM is a key regulator for signaling pathways involving SH3 domain-containing membrane proteins, e.g., the Src
family of kinases, since the QM protein directly interacts with the SH3 domain (37
). Moreover, the yeast QM homologue, GRC5, is involved in translational control of gene expression in S. cerevisiae
, based on the observation that GRC5 directly participates in the recombination of 60S and 40S ribosomal protein subunits (37
). Several QM homologues have been identified in plants, but their physiological functions have not yet been described.
Our objectives in this study were to examine the cellular consequences of endogenous manipulation of intracellular proline levels, particularly under conditions of oxidative stress. Since proline supplementation is cytoprotective to fungal cells (including yeast), we developed a conditional life/death in yeast to identify plant gene products that protect cells with reduced proline levels subjected to oxidative stress. In this report we show that modulation of intracellular proline is effective in ameliorating lethal levels of oxidative stress. Moreover, we describe a novel tomato gene (tQM) that rescues yeast strains with limited proline levels from a variety of ROS-inducing stimuli. Taken together, our data further demonstrate the importance of proline in stress tolerance.