ciferase and the 5 UTRs of plant mRNA for the ribosomal protein S15 and phyAdo not compete in the
binding assays (Xiang and Oliver, unpublished results).
More important, the in vitro binding of this factor(s) is sensitive to the GSH/GSSG ratio, a
parameter that, in turn, is dependent on the H 2 O 2 concentration. Inclusion of 5 mM GSSG in the assay
reaction completely abolished the binding, and dithiothreitol (DTT) and GSH enhanced the binding.
However, H 2 O 2 did not significantly alter the binding, indicating that the effect of H 2 O 2 might be
indirect through changing the GSH/GSSG ratio (Xiang and Oliver, unpublished results). These results
suggest that at high GSH/GSSG ratios, as would be found in unstressed plant cells, the factor binds
to the 5 UTR of GSH1mRNA and restricts the de novo synthesis of -ECS, therefore preventing
the synthesis of excessive GSH that is not needed. In contrast, at low GSH/GSSG ratios, as would be
found following H 2 O 2 or Cu^2 treatments or under oxidative stress, the binding of the factor decreases,
derepressing the translation of GSH1mRNA. Subsequently, de novo synthesis of -ECS would
increase, and as a result, more GSH would be synthesized to replenish the reduced GSH pool within
cells.
This is consistent with the observation of the oxidative stress–stimulated accumulation of the steady-
state -ECS protein level in CuCl 2 -treatedArabidopsistissues. Cu^2 is known to decrease the
GSH/GSSG ratio and to cause oxidative stress as well as to increase the GSH1transcript level in our sys-
tem [20].
- Significance of Translational Control by Oxidative Stress
Translational control is the regulation of the efficiency of translation of mRNAs, either globally on gen-
eral protein synthesis or selectively on a specific mRNA or a subset of mRNAs. Both activation and re-
pression of mRNA translation occur very quickly as compared with transcriptional regulation, thus af-
fording cells directness, rapidity, and reversibility in regulating gene expression in response to constantly
changing living conditions [30]. Numerous studies have indicated translational control of individual or
suites of genes in plants (for review, see Ref. 31, 32, and 33). But there are very few cases in which the
molecular mechanism is understood. In higher plants, adaptive responses to oxidative stress are
widespread and crucial for plant survival and crop productivity. However, translational control has not
been well explored in these responses.
The translational control of GSH1mRNA by oxidative stress is significant in that the specific RNA-
binding complex acts as both an oxidative stress sensor and a modulator of translation. The GSH/GSSG
ratio would serve as a signal of oxidative stress and a switch to regulate GSH synthesis by modulating the
translation of GSH1mRNA. It is interesting to speculate that translational control by GSH/GSSG may
regulate the expression of a suite of enzymes whose activity is modulated by oxidative stress. GSH re-
ductase (GR) expression appears to be translationally regulated in maize bundle sheath cells [34]. Al-
though there is GR mRNA in this tissue, there is little or no GR protein. These authors propose that the
cellular redox potential possibly mediated through the NADP/NADPH ratio controls the translation of
GR mRNA. Thus it is possible that cellular redox status may control the translation of a suite of proteins
involved in protecting plants from oxidative stress.
C. Metabolic Regulation
- Feedback Inhibition of -EC Synthetase by GSH in Vivo
The role of feedback inhibition by GSH on -EC synthetase has been extrapolated from in vitro results
[14]. Whether this feedback inhibition operates in vivo in higher plants has not been verified. A molecu-
lar genetic approach was taken to assess whether the feedback inhibition of -ECS by GSH functions in
vivo.
The transgenic Arabidopsisplants overexpressing Arabidopsis -ECS indicate that large increases
in -ECS protein level do not necessarily result in parallel increases in GSH level (Xiang and Oliver, un-
published results). Similar results were reported for poplar plants overexpressing Escherichia coli -ECS
[35]. These observations are indicative of metabolic regulation of -ECS activity in vivo, most likely the
feedback inhibition by GSH. Another way to demonstrate whether such regulation operates in vivo is to
examine the metabolic flux through the -ECS–catalyzed reaction in the plant tissues with lowered GSH
levels.
542 XIANG AND OLIVER