Maturation of Arabidopsis Seeds Is Dependent on Glutathione Biosynthesis within the Embryo  

Abstract

Glutathione (GSH) has been implicated in maintaining the cell cycle within plant meristems and protecting proteins during seed dehydration. To assess the role of GSH during development of Arabidopsis (Arabidopsis thaliana [L.] Heynh.) embryos, we characterized T-DNA insertion mutants of GSH1, encoding the first enzyme of GSH biosynthesis, g-glutamyl-cysteine synthetase. These gsh1 mutants confer a recessive embryo-lethal phenotype, in contrast to the previously described GSH1 mutant, root meristemless 1(rml1), which is able to germinate, but is deficient in postembryonic root development. Homozygous mutant embryos show normal morphogenesis until the seed maturation stage. The only visible phenotype in comparison to wild type was progressive bleaching of the mutant embryos from the torpedo stage onward. Confocal imaging of GSH in isolated mutant and wild-type embryos after fluorescent labeling with monochlorobimane detected residual amounts of GSH in rml1 embryos. In contrast, gsh1 T-DNA insertion mutant embryos could not be labeled with monochlorobimane from the torpedo stage onward, indicating the absence of GSH. By using high-performance liquid chromatography, however, GSH was detected in extracts of mutant ovules and imaging of intact ovules revealed a high concentration of GSH in the funiculus, within the phloem unloading zone, and in the outer integument. The observation of high GSH in the funiculus is consistent with a high GSH1-promoter::b-glucuronidase reporter activity in this tissue. Development of mutant embryos could be partially rescued by exogenous GSH in vitro. These data show that at least a small amount of GSH synthesized autonomously within the developing embryo is essential for embryo development and proper seed maturation.

Inactivation of Thioredoxin Reductases Reveals a Complex Interplay between Thioredoxin and Glutathione Pathways in Arabidopsis Development

“…Even ntra ntrb rml1 homozygous seeds are able to sustain some growth on a glutathione medium, showing that their viability is not impaired. This notion was recently contradicted by the characterization of a knockout mutant in the GSH1 gene in which no seed formation takes place (Cairns et al, 2006). This mutant revealed that a small amount of GSH is necessary for embryo development.…”

The NADPH-dependent thioredoxin system constitutes a functional backup for cytosolic glutathione reductase in Arabidopsis

“…Antisense lines for Arabidopsis GSH1 indicated that 5% of wild-type GSH, which would result in an E GSH of ϷϪ240 mV, already limits the growth rate and renders plants more sensitive to environmental stress (40). Progressive growth inhibition and increasing stress sensitivity is also apparent for the even more severely affected rml1 mutant, which has Ͻ5% of wild-type GSH, and embryo lethal gsh1 deletion mutants (41,42). Deletion of GR and a concomitant 21-fold increase in GSSG have been reported to cause oxidation of GRXs in S. cerevisiae (20).…”

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

“…Although MCB preferentially labels GSH, the interpretation of images is complicated by vacuolar sequestration of GSH-bimane conjugates (22,23). In addition, leaf cells can be difficult to load with the dye because of limited permeability of the leaf cuticle and the need for vacuum infiltration of leaf fragments.…”