sponsive to heavy metals and JA and possibly other ciselements to be identified in the promoter (Xiang
and Oliver, unpublished data). JA and heavy metals appear to use parallel signaling pathways that even-
tually converge on a small region in the GSH1promoter.
At the translational level, the translation efficiency of the mRNAs produced by increased tran-
scription may be altered in response to the cellular redox state. In unstressed cells, the mRNA could be
repressed by a protein factor(s) present in the cytosol that binds to the 5 UTR of these mRNAs (at least
to the mRNA for GSH1) and prevents their translation. Oxidative stress–generated ROS as well as
heavy metals and xenobiotics decrease the GSH/GSSG ratio and cause this protein to dissociate from
the mRNA molecules. Once this protein has dissociated, the mRNAs are translated and the rate-limit-
ing enzyme for GSH synthesis is produced. This binding activity may serve as both a redox sensor and
a modulator for the translation of GSH1mRNA in accordance with oxidative stress that the plants are
experiencing. This translation control model is consistent with the observation that neither GSH, GSSG,
nor H 2 O 2 affected the transcript level of GSH metabolic genes [20]. Using this model, it can be better
interpreted that translational control is functioning in a more direct and more rapid response to the fluc-
tuating GSH/GSSG ratio in plant cells when compared with the much slower response through tran-
scriptional control.
Given the fine metabolic control mechanisms (feedback inhibition and substrate availability) and
possible posttranslational modification of GSH metabolic enzymes [18], plant cells have evolved multi-
level regulatory mechanisms for GSH homeostasis. The significance of the multilevel regulation model
is severalfold. First, it enables both long-term (transcriptional control) and short-term (translational) re-
sponses to the fluctuating oxidative stress status. Second, the translational control affords cells re-
versibility and flexibility in response to the GSH/GSSG ratio, an indicator of oxidative stress status.
Third, the redox-sensitive nature of the RNA-binding complex is particularly important in that it may act
as an oxidative stress sensor modulating the rate-limiting enzyme level for GSH synthesis and ensuring
that an optimal GSH/GSSG ratio is maintained. It is likely that other genes in this pathway are also reg-
ulated by this regulatory mechanism. Sessile higher plants have evolved sophisticated oxidative stress
sensing and modulating mechanisms that give plant cells the plasticity required for their adaptive re-
sponses in a constantly changing environment.
In addition, GSH synthesis coordinates with cysteine synthesis and utilization of GSH. The coordi-
nation between GSH synthesis and cysteine formation is also reflected metabolically. Sulfate uptake and
assimilation are thought to be controlled by GSH [44,45]. As the storage form of organic sulfur, GSH has
to be transported from source tissues to sink tissues and degraded in a controlled manner to release cys-
teine for protein synthesis and other metabolic utilization. These processes further increase the complex-
ity of GSH homeostasis at the whole plant level.
V. PERSPECTIVES
Taken together, these results demonstrate that the expression of -ECS is subject to multilevel regulation
and indicate that GSH homeostasis at the whole plant level is very complex. Temporal multilevel regula-
tion is further complicated with spatial control mechanisms and transport within the plant body. It is in-
teresting and challenging to pursue how GSH synthesis and transport are regulated and coordinated
among different organs and tissues at the whole plant level.
Preliminary results have shown that the genes encoding the rate-limiting enzymes for both pathways
are coordinated at least at the transcript level. It is expected that these two pathways are simultaneously
activated in response to environmental stresses where large consumption of GSH takes place. It has been
demonstrated that in response to heavy metal exposure, both cysteine synthesis and GSH synthesis are el-
evated in Arabidopsisplants (Xiang and Oliver, unpublished results). Future investigation is also required
to demonstrate at the molecular level the coordination between sulfate uptake and GSH synthesis.
Elucidation of the RNA-binding complex of the 5 UTR of GSH1mRNA should shed light on the
molecular mechanisms by which oxidative stress controls the synthesis of GSH. An integrated approach
combining molecular, biochemical, and genetic means has been taken to identify the components of the
RNA-binding complex. Once the identity of the binding complex is resolved, other interesting questions,
such as whether this binding complex regulates other mRNA species, especially oxidative stress-related
transcripts, can be addressed.
546 XIANG AND OLIVER