Characterization of the extracellular γ‐glutamyl transpeptidases, GGT1 and GGT2, in Arabidopsis

Abstract

gamma-Glutamyl transpeptidase (GGT) is the only enzyme known that can cleave the gamma-peptide bond between glutamate and cysteine in glutathione, and is therefore a key step in glutathione degradation. There are three functional GGT genes in Arabidopsis, two of which are considered here. GGT1 and GGT2 are apoplastic, associated with the plasma membrane and/or cell wall. RNA blots and analysis of enzyme activity in knockout mutants suggest that GGT1 is expressed most strongly in leaves but is found throughout the plant. A GGT1::GUS fusion construct showed expression only in vascular tissue, specifically the phloem of the mid-rib and minor veins of leaves, roots and flowers. This localization was confirmed in leaves by laser microdissection. GGT2 expression is limited to embryo, endosperm, outer integument, and a small portion of the funiculus in developing siliques. The ggt2 mutants had no detectable phenotype, while the ggt1 knockouts were smaller and flowered sooner than wild-type. In ggt1 plants, the cotyledons and older leaves yellowed early, and GSSG, the oxidized form of glutathione, accumulated in the apoplastic space. These observations suggest that GGT1 is important in preventing oxidative stress by metabolizing extracellular GSSG, while GGT2 might be important in transporting glutathione into developing seeds.

Aγ-Glutamyl Transpeptidase-Independent Pathway of Glutathione Catabolism to Glutamate via 5-Oxoproline in Arabidopsis

“…The lack of involvement of GGT as a major component of GSH metabolism agrees with the observations in our GGT knockout plants, where no significant changes in GSH levels were measured in any tissues except in the apoplast (Ohkama-Ohtsu et al, 2007a). While some GSH metabolism occurs through the GGT enzymes in plants and this metabolism is physiologically important in plants, it is clearly a minor portion of total GSH degradation.…”

“…Since GGT2 is only expressed in young siliques (Ohkama-Ohtsu et al, 2007a), it was not included in this study. As shown in Supplemental Table S1 and our previous reports (Ohkama-Ohtsu et al, 2007a, 2007b, GGT activity in the leaves from ggt1 and ggt4 plants was about 10% and 90% of wild-type GGT activity, respectively, while the ggt1/ggt4 double mutant had no detectable GGT activity.…”

“…Since GGT2 is only expressed in young siliques (Ohkama-Ohtsu et al, 2007a), it was not included in this study. As shown in Supplemental Table S1 and our previous reports (Ohkama-Ohtsu et al, 2007a, 2007b, GGT activity in the leaves from ggt1 and ggt4 plants was about 10% and 90% of wild-type GGT activity, respectively, while the ggt1/ggt4 double mutant had no detectable GGT activity. As the ggt1-1 and ggt4-1 plants are in the Landsberg background (Ohkama-Ohtsu et al, 2007a, 2007b and oxp1-1 is in Columbia, oxp1-1 was crossed to Landsberg wild-type plants, and a mixture of F3 seeds derived from five F2 plants carrying the homozygous oxp1-1 mutation was used as a control.…”

“…All of the enzymes in this cycle are reported in plants except the Cys-Gly dipeptidase. Soluble and bound GGT activity has been detected in tomato (Solanum lycopersicum), onion (Allium cepa), radish (Raphanus sativus), and Arabidopsis (Martin and Slovin, 2000;Storozhenko et al, 2002;Nakano et al, 2004;Shaw et al, 2005;Grzam et al, 2007;Martin et al, 2007;Ohkama-Ohtsu et al, 2007a, 2007b. Recently, we analyzed the functions of Arabidopsis GGT proteins using knockout mutants (Ohkama-Ohtsu et al, 2007a, 2007b.…”

“…GGT4 (At4g29210) was originally called GGT 3 in our earlier papers (Ohkama-Ohtsu et al, 2007a, 2007b but was renamed here to agree with the nomenclature of Martin et al (2007), who simultaneously discovered it function (Grzam et al, 2007). GGT1 mitigates oxidative stress by degrading oxidized GSH in the extracellular space, and GGT2 (also found in the apoplast) may be involved in GSH transport into siliques (Ohkama-Ohtsu et al, 2007a). GGT4 is located in the vacuole, where it is responsible for the degradation of GSH conjugates formed by glutathione S-transferase reactions (Ohkama-Ohtsu et al, 2007b).…”