Multilevel regulation of GSH synthesis and homeostasis has been implicated in a number of studies
[18–20]. To date, at least five levels of control of steady-state GSH concentrations have been identified
or implicated. These are (1) control of the transcription of the genes for GSH synthesis, (2) posttrans-
critional regulations including translational controls, (3) feedback inhibition of GSH formation at -EC
synthetase, (4) rate limitation of GSH synthesis by -EC synthetase activity, and (5) substrate availabil-
ity. These temporal regulatory mechanisms are further complicated by spatial regulations at the whole
plant level and GSH transport and turnover.
A. Transcriptional Regulation
- The Genes for GSH Synthesis and Recycling Are Coordinately Up-Regulated in
Response to Heavy Metals and Jasmonic Acid
It was previously demonstrated that Arabidopsisplants treated with cadmium or copper responded by in-
creasing transcription of the genes for GSH synthesis, -ECS and GSH synthetase, as well as GSH re-
ductase [20]. The response was specific for the metals whose toxicity is mitigated through phytochelatins.
Other toxic and nontoxic metals did not alter messenger RNA (mRNA) levels. Feeding experiments sug-
gested that neither oxidative stress, as results from exposure to H 2 O 2 , nor oxidized or reduced glutathione
levels were responsible for activating transcription of these genes.
Jasmonic acid (JA), a naturally occurring growth regulator that has important roles in plant devel-
opment and in insect and disease resistance (for review see Refs. 21 and 22), also activated the same suite
of genes. This suggests that JA may be a general stress signal molecule and may be involved in the sig-
nal transduction pathway for copper and cadmium.
The up-regulation of the transcripts by JA and heavy metals was dependent on de novo protein syn-
thesis. The elevated transcript accumulation was transcriptionally controlled. Interestingly, these genes
respond to heavy metals and JA in a coordinated manner [20].
- The Responsiveness to Heavy Metals and JA Appears to Be Mediated by an
as-1–Like Element in the GSH1 Promoter
Part of the complexity in understanding the regulation of -ECS activity is that the transcription of GSH1
is regulated by both heavy metals and JA. An integrated approach was taken to investigate the molecular
mechanisms by which JA and heavy metals control the expression of GSH1. Promoter deletion analysis
was conducted to locate the heavy metal– and JA-responsive ciselements in the GSH1promoter.Ara-
bidopsisgenomic clones for GSH1were isolated. The GSH1promoter and its 5 coding region were se-
quenced. Promoter deletion analysis for GSH1identified a region responsive to JA and heavy metals. An
apparentas-1–type element is located within this region. Removal of this element renders the promoter
nonresponsive to JA or heavy metals (C. Xiang and D. J. Oliver, unpublished results). The as-1–type el-
ements are known to respond to multiple external stimuli, including JA and heavy metals [23,24]. This
as-1–type element in the GSH1promoter may be responsible for the up-regulation by JA and heavy met-
als. It is speculated that other genes regulated coordinately with GSH1may also possess as-1–like ele-
ments in their promoters.
- Heavy Metal and JA Signaling Pathways Are Parallel
How does the GSH1promoter respond to both JA and heavy metals? One interpretation of the data
is that JA is an intermediate in the heavy metal signal transduction pathway. To ascertain whether
JA and heavy metals share a common signal transduction pathway in the activation of GSH1or whether
there are two different pathways that converge on a common ciselement, two Arabidopsismutants,
jar1andfad3-2fad7-2fad8 (18:3), were analyzed. The mutant jar1is JA unresponsive [25] and
the triple mutant fad3-2fad7-2fad8is deficient in the jasmonate precursor, linolenic acid [26]. RNA gel
blot analysis demonstrates that heavy metal signaling and JA signaling pathways are independent of
each other. In both the JA-deficient triple mutant and jar1, the GSH1gene is still able to respond to
heavy metal treatment in the same way as wild-type plants (Xiang and Oliver, unpublished results).
Clearly, JA is not a mandatory intermediate in the heavy metal response pathway. However, these
results cannot rule out the possibility that the exposure to heavy metals triggers the production of JA
in vivo.
540 XIANG AND OLIVER