Unbalanced activation of glutathione metabolic pathways suggests potential involvement in plant defense against the gall midge Mayetiola destructor in wheat

Abstract

Glutathione, γ-glutamylcysteinylglycine, exists abundantly in nearly all organisms. Glutathione participates in various physiological processes involved in redox reactions by serving as an electron donor/acceptor. We found that the abundance of total glutathione increased up to 60% in resistant wheat plants within 72 hours following attack by the gall midge Mayetiola destructor, the Hessian fly. The increase in total glutathione abundance, however, is coupled with an unbalanced activation of glutathione metabolic pathways. The activity and transcript abundance of glutathione peroxidases, which convert reduced glutathione (GSH) to oxidized glutathione (GSSG), increased in infested resistant plants. However, the enzymatic activity and transcript abundance of glutathione reductases, which convert GSSG back to GSH, did not change. This unbalanced regulation of the glutathione oxidation/reduction cycle indicates the existence of an alternative pathway to regenerate GSH from GSSG to maintain a stable GSSG/GSH ratio. Our data suggest the possibility that GSSG is transported from cytosol to apoplast to serve as an oxidant for class III peroxidases to generate reactive oxygen species for plant defense against Hessian fly larvae. Our results provide a foundation for elucidating the molecular processes involved in glutathione-mediated plant resistance to Hessian fly and potentially other pests as well.

Figure 1. Hessian fly induces higher levels of glutathione in wheat tissues at the feeding site in resistant plants, but did not affect the ratio of GSH/GSSG.

Black and grey bars represent data from susceptible (Newton) and resistant (Molly) plants, respectively. GSH+GSSG represent changes in the abundance of total glutathione, whereas GSSG represents changes in abundance of oxidized glutathione. The bottom panel represents the ratio of GSSG/GSH.

Figure 2. Hessian fly induces higher levels of enzymatic activity and transcript abundance of glutathione synthetases.

Black and grey bars represent data from susceptible (Newton) and resistant (Molly) plants, respectively. (a) Percentage change in enzymatic activity of glutathione synthetases in plants at different time points after Hessian fly infestation. (b) Fold changes of transcript abundance determined by qPCR using the primer pair common to CK156077, AJ579381 and AJ579382. (c) Fold changes of transcript abundance determined by qPCR using the primer pair specific to AJ579380.

Figure 3. Hessian fly induces higher levels of enzymatic activity and transcript abundance of glutathione peroxidases in both resistant and susceptible plants.

Black and grey bars represent data from susceptible (Newton) and resistant (Molly) plants, respectively. (a) Percentage change in enzymatic activity of glutathione peroxidases in plants at different time points after Hessian fly infestation. (b) Fold changes of transcript abundance determined by qPCR using the primer pair specific to BJ254939. (c) Fold changes of transcript abundance determined by qPCR using the primer pair specific to AY364468.

Figure 4. Hessian fly induces higher levels of enzymatic activity and transcript abundance of glutathione reductases in susceptible plants, but not in resistant plants.

Black and grey bars represent data from susceptible (Newton) and resistant (Molly) plants, respectively. (a) Percentage change in enzymatic activity of glutathione reductases in plants at different time points after Hessian fly infestation. (b) Fold changes of transcript abundance determined by qPCR using the primer pair specific to AY364467. (c) Fold changes of transcript abundance determined by qPCR using the primer pair specific to FK827496.

Figure 5. Hessian fly induces higher levels of transcript abundance of genes encoding glutathione degradation enzymes in susceptible plants, but not in resistant plants.

Black and grey bars represent data from susceptible (Newton) and resistant (Molly) plants, respectively. qPCR was carried out using primers specific to the γ-glutamyltransferase genes BU100842 (GT42) and AK333876 (GT76), and to the tripeptide aminopeptidase genes CJ717454 (TPA54) and HX136475 (TPA75), respectively.

Figure 6. Metabolic pathways of glutathione synthesis, recycling, detoxification, and degradation; and a model for glutathione to serve as an oxidant for class III peroxidases during generation of ROS such as hydrogen peroxides.

Glu, Cys, γ-EC, Gly, and Cys-Gly represent glutamate, cysteine, γ-glutamylcysteine, glycine, and cysteinylglycine, respectively. γ-GCS, GS, GT, TPA, GST, GR, GPx, and III-Px represent γ-glutamylcysteine synthetases, glutathione synthetases, γ-glutamyltransferases, tripeptide aminopeptidases, glutathione S-transferases, glutathione reductases, glutathione peroxidases, and class III peroxidases, respectively. Blue arrows indicate direction of metabolite flow, whereas yellow arrows indicate transport of GSSG and GSH between cytosol and apoplast, where GSSG serves as an oxidant for class III peroxidases during ROS generation.