Plant homologs of the Plasmodium falciparum chloroquine-resistance transporter, Pf CRT, are required for glutathione homeostasis and stress responses

Abstract

In Arabidopsis thaliana, biosynthesis of the essential thiol antioxidant, glutathione (GSH), is plastid-regulated, but many GSH functions, including heavy metal detoxification and plant defense activation, depend on cytosolic GSH. This finding suggests that plastid and cytosol thiol pools are closely integrated and we show that in Arabidopsis this integration requires a family of three plastid thiol transporters homologous to the Plasmodium falciparum chloroquine-resistance transporter, PfCRT. Arabidopsis mutants lacking these transporters are heavy metal-sensitive, GSH-deficient, and hypersensitive to Phytophthora infection, confirming a direct requirement for correct GSH homeostasis in defense responses. Compartment-specific measurements of the glutathione redox potential using redox-sensitive GFP showed that knockout of the entire transporter family resulted in a more oxidized glutathione redox potential in the cytosol, but not in the plastids, indicating the GSH-deficient phenotype is restricted to the cytosolic compartment. Expression of the transporters in Xenopus oocytes confirmed that each can mediate GSH uptake. We conclude that these transporters play a significant role in regulating GSH levels and the redox potential of the cytosol.T he potentially damaging end-products of aerobic energy metabolism, reactive oxygen species (ROS), are powerful signaling components linking growth, metabolism, and defense responses in cells (1-4). In plant cells, a complex antioxidant network with glutathione (GSH) at its center has evolved to buffer ROS. Because both the levels and oxidation state of GSH are directly influenced by ROS, GSH is a key redox-signaling component (5-10).GSH is synthesized in two steps (11) catalyzed by the ratelimiting glutamate-cysteine ligase (GSH1; EC 6.3.2.2) and glutathione synthase (GSH2; EC 6.3.2.3). In Arabidopsis, GSH1 is exclusively targeted to the plastid, while GSH2 is targeted to both plastid and cytosol (12). Consequently, the pathway intermediate, γ-glutamylcysteine (γ-EC), must be exported from the plastid to allow for cytosolic GSH biosynthesis. This finding was recently confirmed by the observation that inviable gsh2 mutants can be fully complemented by expression of functional GSH2 only in the cytosol (13), suggesting that thiol transport between compartments is essential for maintaining both GSH levels and redox-based signaling pathways, although no plastid thiol transporters have yet been identified (13-17).

The Involvement of Hydrogen-producing and ATP-dependent NADPH-consuming Pathways in Setting the Redox Poise in the Chloroplast of Chlamydomonas reinhardtii in Anoxia

“…With midpoint potentials of Ϫ420 mV (54), Ϫ345 mV (55), and Ϫ320 mV and stoichiometries relative to PSI of 4, 2, and 5 for ferredoxin, FNR, and NADP ϩ , respectively (56 -58 and references therein), this translates, assuming that equilibrium is achieved in the dark, to an ambient redox potential of approximately Ϫ370 mV after a few minutes of anoxia. This is only mildly more reducing than the redox poise Ϫ320 mV reported in the plastid of epidermal cells in A. thaliana (59) and hardly less than the estimate made previously (29) of the steady redox poise in the stroma of C. reinhardtii in the light in anoxia.…”

Cytosolic and Chloroplastic DHARs Cooperate in Oxidative Stress-Driven Activation of the Salicylic Acid Pathway

“…9) suggests that the status of this key cellular thiol/disulfide compound also may be an important factor. If so, this would add to data showing that SA-related signaling is compromised in mutants for chloroplast-cytosol glutathione exchange (Maughan et al, 2010) and in cat2 lines in which glutathione synthesis is partially blocked (Han et al, 2013a). It also provides further evidence that the activation of H 2 O 2 -triggered signaling inside the cell depends on a secondary modulation of antioxidant status, notably that of glutathione.…”

“…In any case, the ability of chloroplastic DHAR3 to complement the quadruple mutant raises questions about the potential importance of redox exchange across the inner envelope membrane. Chloroplasts have long been known to be competent in GSSG and DHA uptake (Anderson et al, 1983), and more recently, chloroplast envelope glutathione and ascorbate transporters have been identified in Arabidopsis (Maughan et al, 2010;Miyaji et al, 2015).…”

Intracellular Redox Compartmentation and ROS-Related Communication in Regulation and Signaling

“…Nevertheless, work over recent decades has demonstrated the uptake of ascorbate and glutathione into cells or subcellular organelles at rates well in excess of diffusion. Because of space limitations, we refer the reader to studies cited in other recent articles (Maughan et al, 2010;Noctor et al, 2013;Szarka et al, 2013;Fernie and Tóth, 2015;Foyer, 2015). With the exceptions noted below, many of the transporters remain to be characterized at the molecular level.…”