rescence stems, where less -ECS protein is present. These data again indicate the multilevel regula-
tion of GSH synthesis.
Intriguingly, GSH levels on a fresh weight basis vary significantly from tissue to tissue. It appears
that reproductive organs (flower buds, flowers, and siliques) have higher GSH levels than other organs,
although the transcript levels for GSH1are low in flower buds and flowers. Leaves, the source tissue, are
generally thought to be the major site of GSH synthesis; however, they have the lowest GSH level among
all tissues. The differences in GSH levels in tissues may indicate the intrinsic differences in GSH re-
quirements of different organs and active GSH transport among these organs.
- Transport and Turnover of GSH
Transport of GSH in higher plants, both intracellular and long-distance interorgan, is another important
level of regulation of GSH homeostasis. Up to now, the existence of intracellular GSH transporters has
not been demonstrated. Although significant progress has been made on the regulation of sulfur uptake
and assimilation by GSH [44,45] and GSH transport between organs has been studied [46] the regulation
of long-distance interorgan GSH transport deserves further investigation in greater detail.
One of the biological functions assigned to GSH is the storage and transport of reduced sulfur (cys-
teine). Therefore, GSH has to be degraded in order to make cysteine available for protein synthesis and
other utilization. Unfortunately, very little is known about the regulation of GSH degradation in higher
plants, although the pathway(s) is known and similar to that in animals [47–50]. The DNA sequence in-
formation for -glutamyl transpeptidase and 5-oxo-L-prolinase is available from GenBank. The 5-oxo-L-
prolinase is extremely conserved through evolution in that its more than 3000 bp coding sequence is in-
tronless. This single open reading frame–encoded polypeptide shares high amino acid sequence
homology with that in other organisms from prokaryotes to human. Cloning of the genes encoding the
key enzymes of the GSH degradation pathway will provide necessary tools to probe the regulation mech-
anisms for GSH breakdown.
III. COORDINATION BETWEEN GSH AND CYSTEINE SYNTHESIS
Cysteine is a key substrate for GSH synthesis. It has been expected that there should be coordination be-
tween sulfur uptake/assimilation and GSH synthesis [5]. Indeed, the transcription of genes for GSH syn-
thesis ( -ECS) and for sulfur assimilation (5 -adenylylsulfate [APS] reductases) is coordinated in re-
sponse to heavy metals in plants [20,51]. The expression of mRNA and enzyme activity for the sulfur
assimilation enzyme APS reductase was analyzed in Brassica junceaexposed to CdSO 4. In plants ex-
posed to varying Cd doses up to 200 M the level of APS reductase mRNA increased linearly with the
dose but the activity lagged behind the mRNA and was inhibited above 50 M. When the plants were
treated with Zn, Pb, Cu, and Hg, APS reductase mRNA and enzyme activity increased synchronously
[51]. Figure 1 shows that the expression of APRgenes encoding the rate-limiting enzyme, APS reductase,
for cysteine synthesis coordinates with that of GSH1for GSH synthesis in response to JA treatment.
GSH1and two APRgenes analyzed in this experiment were induced in 1 hr by JA treatment. The induc-
tion kinetics of the APRgenes was similar to that of GSH1. The induction by JA appears to be dose de-
pendent. Unlike GSH1, the APRgenes are strongly responsive to H 2 O 2.
These results suggest that the genes encoding the rate-limiting enzymes for both pathways are coor-
dinately regulated in response to environmental stresses. The cross talk between sulfur uptake/assimila-
tion and GSH synthesis is currently under investigation.
IV. MULTILEVEL REGULATION OF GSH HOMEOSTASIS IN
RESPONSE TO ENVIRONMENTAL STRESSA working model for the multilevel regulation of GSH1expression and GSH homeostasis based on the
experimental results is illustrated in Figure 2.
At the transcriptional level, heavy metals and JA up-regulate the transcription of the genes for GSH
synthesis. The transcriptional control of GSH1expression in response to heavy metals and JA is mediated
by the ciselements that have been recruited by GSH1promoter. These include the as-1–type element re-
544 XIANG AND OLIVER