Molecular Mechanisms of Proline-Mediated Tolerance to Toxic Heavy Metals in Transgenic Microalgae

Abstract

Pro has been shown to play an important role in ameliorating environmental stress in plants and microorganisms, including heavy metal stress. Here, we describe the effects of the expression of a mothbean delta(1)-pyrroline-5-carboxylate synthetase (P5CS) gene in the green microalga Chlamydomonas reinhardtii. We show that transgenic algae expressing the mothbean P5CS gene have 80% higher free-Pro levels than wild-type cells, grow more rapidly in toxic Cd concentrations (100 microM), and bind fourfold more Cd than wild-type cells. In addition, Cd-K edge extended x-ray absorption fine structure studies indicated that Cd does not bind to free Pro in transgenic algae with increased Pro levels but is coordinated tetrahedrally by sulfur of phytochelatin. In contrast to P5CS-expressing cells, Cd is coordinated tetrahedrally by two oxygen and two sulfur atoms in wild-type cells. Measurements of reduced/oxidized GSH ratios and analyses of levels of malondialdehyde, a product of the free radical damage of lipids, indicate that free Pro levels are correlated with the GSH redox state and malondialdehyde levels in heavy metal-treated algae. These results suggest that the free Pro likely acts as an antioxidant in Cd-stressed cells. The resulting increased GSH levels facilitate increased phytochelatin synthesis and sequestration of Cd, because GSH-heavy metal adducts are the substrates for phytochelatin synthase.

Up-regulation of antioxidant and glyoxalase systems by exogenous glycinebetaine and proline in mung bean confer tolerance to cadmium stress

“…5a & b), suggesting that both proline and betaine protect against Cd-dependent oxidative damage by enhancing antioxidant defense and MG detoxification systems (Demiral and Türkan 2004;Okuma et al 2004;Park et al 2006;Huang et al 2009;Hossain and Fujita 2010). Similar findings have been reported by other researchers demonstrating higher Cd tolerance by in vivo betaine or proline synthesis in plants (Siripornadulsil et al 2002;Shao et al 2008). …”

“…An increase in GSH content due to proline pretreatment and lower lipid peroxidation has recently been reported (Xu et al 2009). Transgenic plants for higher proline and betaine biosynthesis also showed higher GSH, GSH/GSSG ratio and lower GSSG content as compared to wild-type under Cd medium and other stress conditions (Siripornadulsil et al 2002;Yang et al 2007). Therefore, both proline and betaine might play a significant role in maintaining higher GSH level either through efficient recycling or by modulating higher GSH synthesis (Kocsy et al 2005;Hossain and Fujita 2010).…”

“…However, both proline and betaine-treated seedlings could partially alleviate the stress-induced oxidative damage by maintaining higher Gly I and Gly II activities suggesting that both of them were able to enhance GSH regeneration and glutathione redox state via the glyoxalase system. The high GSH levels in turn facilitate phytochelatin synthesis and sequestration of heavy metal phytochelatin conjugates in the vacuole (Siripornadulsil et al 2002).…”

Proline modulates the intracellular redox environment and protects mammalian cells against oxidative stress

“…We also observed that proline preserves the intracellular glutathione pool, which is the major redox buffer of the cell. Proline protection of GSH was previously observed in Chlamydomonas reinhardtii exposed to toxic Cd levels [60]. Thus, proline accumulation appears to be an effective means for protecting the glutathione redox state of the cell under stress conditions perhaps by directly scavenging ROS.…”

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“…Acetyl-CoA carboxylase (ACC) catalyzes the biotin-dependent carboxylation of acetyl-CoA to produce malonyl-CoA. This is the first and the committed step in the biosynthesis of long-chain fatty acids [10].…”