In order to dissect the JA and heavy metal signaling pathways, a major effort is currently under way
to isolate Arabidopsismutants deficient in JA or heavy metal responsiveness by using the reporter line
described earlier.
B. Translational Control
- The Translation of GSH1 mRNA Is Regulated in Response to JA and H 2 O 2
The existence of posttranscriptional control for GSH synthesis was demonstrated previously [20]. First,
jasmonic acid treatment increased mRNA levels and the capacity for GSH synthesis but did not alter the
GSH content in unstressed plants, suggesting that posttranscriptional regulation is involved. Second, ox-
idative stress in the form of H 2 O 2 did not up-regulate the transcript level of GSH metabolic genes but did
raise the GSH concentration in plants [27,28] (Xiang and Oliver, unpublished data). These observations
strongly suggest that some additional control mechanisms must be functioning, possibly at the transla-
tional or posttranslational levels. Posttranscriptional regulation of GSH1expression was also implicated
from a study using suspension cell culture [19].
To investigate further the control mechanisms for the expression of GSH1in response to JA and
H 2 O 2 [20], the steady-state transcript and protein levels of the endogenous -ECS were analyzed. It was
found that JA increased the steady-state level of GSH1transcript at least 10-fold but did not significantly
alter the -ECS protein level. In contrast, H 2 O 2 treatment had no appreciable effect on GSH1transcript
level but did elevate the endogenous -ECS protein level after 3 hr or longer exposure (Xiang and Oliver,
unpublished results). This disconnection between the levels of GSH1mRNA and -ECS protein clearly
demonstrates the dramatic change in the translational efficiency of GSH1mRNA or in -ECS protein
turnover in response to H 2 O 2 and JA.
The regulation observed for the endogenous GSH1expression was confirmed for the reporter gene
uidAunder the control of the GSH1gene promoter and its entire 5 untranslated region (5 UTR) in the
transgenicArabidopsisplants. The RNA gel blot analysis of the reporter lines revealed that the transgene
behaved like the endogenous GSH1and the level of GUS mRNA increased following treatment with JA
but not with H 2 O 2. Although the level of GUS mRNA increased with JA treatment, GUS activity as well
as GUS protein level did not significantly change. When the transgenic reporter plants were exposed to
H 2 O 2 , the transcript level remained unchanged, but the GUS protein level increased substantially (Xiang
and Oliver, unpublished results).
Given that these are two unrelated proteins, a change in protein stability for both -ECS and GUS is
unlikely under these conditions. Therefore, the control mechanism uncovered is most likely at the trans-
lational level but not at a posttranslational level. Taken together, these results strongly favor a transla-
tional control mechanism modulating the de novo synthesis of the rate-limiting enzyme for GSH synthe-
sis from the existing GSH1mRNA.
- The Translation of GSH1 mRNA May Be Modulated by a Redox-Sensitive Binding
Activity Specific for the 5 UTR of GSH1 mRNA
The previous observations on the translational control of GSH1mRNA suggest a mechanism that might
be similar to that of the iron-responsive element/iron regulatory proteins (IRE/IRPs)-regulated iron home-
ostasis in animal cells, where cellular iron availability controls the translation of ferritin mRNA [29]. The
IRP binds to the 5 UTR of ferritin mRNA and blocks its translation when the cellular iron concentration
is low. When excess iron is present, IRP dissociates from ferritin mRNA, allowing the synthesis of fer-
ritin, the iron storage protein. JA increased the mRNA level for both -ECS and GUS, but this elevated
transcript level was not paralleled by an increased protein level, suggesting that its translation was
blocked. Exposure to H 2 O 2 increased de novo synthesis of -ECS and GUS from the existing mRNA,
probably by releasing this block on translation. It is speculated that a protein(s) might bind the GSH1
mRNA and the binding of this protein might be altered by H 2 O 2.
Because the GSH1:uidAconstruct contains the 5 UTR but lacks the GSH1coding region and 3 UTR,
this putative ciselement should be within the 5 UTR of the GSH1mRNA. Using RNA gel retardation as-
says and the 5 UTR of GSH1mRNA as a probe, a specific binding activity was identified in protein ex-
tracts from Arabidopsis. This binding activity is specific for the 5 UTR of GSH1mRNA because only
RNA containing the 5 UTR of GSH1mRNA competes for binding with the factor. The RNA for lu-
MULTILEVEL REGULATION OF GLUTATHIONE HOMEOSTASIS 541