Differential targeting of GSH1 and GSH2 is achieved by multiple transcription initiation: implications for the compartmentation of glutathione biosynthesis in the Brassicaceae

Abstract

SummaryThe genome of Arabidopsis thaliana reveals that in this species the enzymes of glutathione biosynthesis, GSH1 and GSH2, are encoded by single genes. In silico analysis predicts proteins with putative plastidic transit peptides (TP) for both genes, but this has not been experimentally verified. Here we report a detailed analysis of the 5¢ends of GSH1 and GSH2 mRNAs and demonstrate the subcellular targeting of the proteins encoded by different transcript types. GSH1 transcript analysis revealed two mRNA populations with short and long 5¢-UTRs, respectively, both including the entire TP sequence. The ratio of long/total GSH1 transcripts was subject to developmental regulation. Transient transformation experiments with reporter gene fusions, bearing long or short 5¢-UTRs, indicated an exclusive targeting of GSH1 to the plastids. Corroborating these results, endogenous and ectopically expressed GSH1 proteins were always present as a single polypeptide species with the size expected for correctly processed GSH1. Finally, the plastidic GSH1 localization was confirmed by immunocytochemistry. Similar to GSH1, multiple transcript populations were found for GSH2. However, here the prevalent shorter transcripts lacked a complete TP sequence. As expected, the large (but less abundant) transcript encoded a plastidic GSH2 protein, whereas GSH2 synthesized from the shorter transcript was targeted to the cytosol. The implications of the results for the compartmentation and regulation of GSH synthesis are discussed.

Transcriptomics of induced defense responses to greenbug aphid feeding in near isogenic wheat lines

“…6C) suggesting increased ascorbate content for ROS detoxification [73]. Also, up-regulation of two transcripts encoding to glutathione synthase 2 (GHS2) involved in glutathione biosynthesis [74] in SB consistent with other antioxidant related transcripts suggest severe feeding-induced stress responses. Elevated expression of antioxidant related transcripts meditating biosynthesis of GSTs, peroxidases, and redox homeostasis in absence of Gb3 in SB suggests basal defense mechanisms have resulted in the production of H 2 O 2 and other toxic compounds in response to greenbug feeding.…”

Subcellular localization and tissue specific expression of amidase 1 from Arabidopsis thaliana

“…To genérate vectors for flúores-cence analysis in transiently transformed plant cells, the expression cassettes containing the genes of the two fluorescent proteins, EGFP and DsRed were excised with Hindllll EcoRl from the vectors pFF19-GFP and pFF19-RFP (Wachter et al 2005), respectively, and integrated into the Hindlll/EcoRl site of the vector pQE-30Xa (Qiagen), which was first modified by XbalINhel restriction followed by religation to eliminate an additional Xbal site, resulting in pSP-EGFP and pSP-DsRed. To créate a control construct (pSP-CRY2(Al-500)-DsRed) for proteins with nuclear localization, a fragment of cryptochrome 2 was amplified which encodes the C-terminal amino acids 501-612.…”

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

“…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.…”