Isoprenylcysteine Methylation and Demethylation RegulateAbscisic Acid Signaling in Arabidopsis W
David H. Huizinga, Olutope Omosegbon, Bilal Omery, and Dring N. Crowell1,2
Department of Biology, Indiana University–Purdue University Indianapolis, Indianapolis, Indiana 46202-5132
Isoprenylated proteins bear an isoprenylcysteine methyl ester at the C terminus. Although isoprenylated proteins have been
implicated in meristem development and negative regulation of abscisic acid (ABA) signaling, the functional role of the
terminal methyl group has not been described. Here, we show that transgenic Arabidopsis thaliana plants overproducing
isoprenylcysteine methyltransferase (ICMT) exhibit ABA insensitivity in stomatal closure and seed germination assays,
establishing ICMT as a negative regulator of ABA signaling. By contrast, transgenic plants overproducing isoprenylcysteine
methylesterase (ICME) exhibit ABA hypersensitivity in stomatal closure and seed germination assays. Thus, ICME is a
positive regulator of ABA signaling. To test the hypothesis that ABA signaling is under feedback regulation at the level of
isoprenylcysteine methylation, we examined the effect of ABA on ICMT and ICME gene expression. Interestingly, ABA
induces ICME gene expression, establishing a positive feedback loop whereby ABA promotes ABA responsiveness of plant
cells via induction of ICME expression, which presumably results in the demethylation and inactivation of isoprenylated
negative regulators of ABA signaling. These results suggest strategies for metabolic engineering of crop species for
drought tolerance by targeted alterations in isoprenylcysteine methylation.
INTRODUCTION
Protein farnesylation is the process by which proteins bearing a
C-terminal CaaXmotif (where C =Cys, a = aliphatic, and X =Met,
Ala, Gln, Ser, or Cys) are posttranslationally modified by the
covalent attachment of a 15-carbon farnesyl group (Clarke,
1992; Zhang and Casey, 1996; Crowell, 2000). This modification
results in the formation of a stable thioether bond between the
Cys of the CaaX motif and the farnesyl moiety, with farnesyl di-
phosphate serving as the farnesyl donor (Figure 1). This lipidation
reaction is catalyzed by protein farnesyltransferase (PFT), which
is a cytosolic enzyme consisting of a- and b-subunits (Clarke,
1992; Zhang and Casey, 1996; Crowell, 2000). In a similar
process, proteins bearing a C-terminal CaaX motif, where X is
Leu or Ile, are modified by the covalent attachment of a 20-
carbon geranylgeranyl group to the Cys of the CaaX motif. This
modification is catalyzed by protein geranylgeranyltransferase
type I (PGGT I), which is also a cytosolic enzyme consisting of an
a-subunit identical to that of PFT and a distinctb-subunit (Clarke,
1992; Zhang and Casey, 1996; Crowell, 2000). A third enzyme,
protein geranylgeranyltransferase type II (PGGT II), also called
RAB geranylgeranyltransferase (RAB GGT), catalyzes the ger-
anylgeranylation of RAB proteins bound to the RAB ESCORT
PROTEIN. All three enzymes have been found in protozoans,
metazoans, fungi, and plants, including pea (Pisum sativum)
(Yang et al., 1993; Qian et al., 1996), tomato (Solanum lycoper-
sicum) (Loraine et al., 1996; Yalovsky et al., 1996), and Arabi-
dopsis thaliana (Cutler et al., 1996; Pei et al., 1998; Caldelari et al.,
2001; Running et al., 2004; Johnson et al., 2005).
In Arabidopsis, a single gene encodes the common a-subunit
of PFT and PGGT I (PLURIPETALA [PLP]; Running et al., 2004), a
second gene encodes the b-subunit of PFT (ENHANCED RE-
SPONSE TO ABA1 [ERA1]; Cutler et al., 1996; Pei et al., 1998),
and a third gene encodes the b-subunit of PGGT I (GERANYL-
GERANYLTRANSFERASE BETA [GGB]; Caldelari et al., 2001;
Johnson et al., 2005). The ERA1 gene was so named because
knockout mutations in this gene cause an enhanced response to
abscisic acid (ABA) in both seed germination and stomatal
closure assays. Consequently, era1 mutants exhibit increased
seed dormancy and stomatal closure in response to ABA and are
drought-tolerant (Cutler et al., 1996; Pei et al., 1998). These
observations suggest that at least one farnesylated protein func-
tions as a negative regulator of ABA signaling. However, to date, a
farnesylated negative regulator of ABA signaling has not been
identified. era1 plants also exhibit enlarged meristems and super-
numerary floral organs, especially petals, and this phenotype is
greatly exaggerated in plpmutants lacking the commona-subunit
ofPFTandPGGT I (Runninget al., 1998, 2004;Bonetta et al., 2000;
Yalovsky et al., 2000; Ziegelhoffer et al., 2000). The more severe
developmental phenotype of plp mutants compared with that of
era1mutants suggests that PGGT I partially compensates for loss
of PFT in era1 plants (Running et al., 2004). This cross-specificity
was later confirmed by the observation that overexpression of the
GGB gene partially suppressed the developmental phenotype of
era1 plants (Johnson et al., 2005). Plants with defects in the GGB
gene exhibit an enhanced response to ABA in guard cells, but not
seeds, and an enhanced response to auxin with respect to lateral
1 Current address: Department of Biological Sciences, Idaho StateUniversity, Pocatello, ID 83209-8007.2 Address correspondence to [email protected] author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) is: Dring N. Crowell([email protected]).WOnline version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.107.053389
This article is a Plant Cell Advance Online Publication. The date of its first appearance online is the official date of publication. The article has been
edited and the authors have corrected proofs, but minor changes could be made before the final version is published. Posting this version online
reduces the time to publication by several weeks.
The Plant Cell Preview, www.aspb.org ã 2008 American Society of Plant Biologists 1 of 15
root formation, but not inhibition of primary root growth (Johnson
et al., 2005). These observations suggest that at least one
geranylgeranylated protein functions as a negative regulator of
ABA signaling and at least one functions as a negative regulator of
auxin signaling. Indeed, ROP2 and ROP6, which are geranylger-
anylated small GTPases (Lemichez et al., 2001; Li et al., 2001),
have been shown to function as negative regulators of ABA
signaling. In addition, AUX2-11 (IAA4), which is a geranylgerany-
lated member of the AUX/IAA family, and ROP2 function as
negative regulators of auxin signaling (Wyatt et al., 1993; Caldelari
et al., 2001; Li et al., 2001), andArabidopsisplants lacking either of
two geranylgeranylatedG-protein g-subunits exhibit an enhanced
response to auxin-induced lateral root formation (Trusov et al.,
2007). Isoprenylated proteins have also been implicated in a
plethora of other processes, such as calcium signal transduction
(Rodrıguez-Concepcion et al., 1999; Xiao et al., 1999), response to
heat and heavymetal stress (Zhu et al., 1993; Dykema et al., 1999;
Suzuki et al., 2002), cytokinin biosynthesis (Galichet et al., 2008),
and regulation of the cell division cycle (Qian et al., 1996;
Hemmerlin and Bach, 1998; Hemmerlin et al., 2000). Given these
multiple roles, it is surprising that, unlike other organisms, Arabi-
dopsisplants lacking detectable PFT and PGGT I activities survive
(Running et al., 2004).
Proteins that are isoprenylated by either PFT or PGGT I are
further modified. First, the aaX portion of the CaaX motif is
proteolytically removed by specific CaaX proteases (STE24 and
FACE-2 in Arabidopsis) (Boyartchuk et al., 1997; Bracha et al.,
2002; Cadinanos et al., 2003); second, the isoprenylcysteine at
Figure 1. Protein Farnesylation, Proteolysis, Methylation, Demethylation, Degradation, and Recycling of the Farnesyl Group.
The transfer of a farnesyl group from farnesyl diphosphate to a CaaX protein is followed by postisoprenylation processing (proteolysis and reversible
methylation). Degradation of the farnesylated protein generates FC, which is oxidized by FC lyase to farnesal. Farnesal is then reduced to farnesol,
which is phosphorylated to farnesyl diphosphate to complete the cycle. Gene names are indicated in italics. CDP, cytidine diphosphate; CTD, cytidine
triphosphate; CHO, aldehyde; OPP, diphosphate; SAH, S-adenosyl-L-homocysteine; SAM, S-adenosyl-L-methionine.
2 of 15 The Plant Cell
the newly formed C terminus is methylated (Figure 1) (Hancock
et al., 1991; Sapperstein et al., 1994; Parish and Rando, 1996;
Parish et al., 1996; Crowell et al., 1998; Bergo et al., 2000;
Rodrıguez-Concepcion et al., 2000). Two distinct isoprenylcys-
teine methyltransferase (ICMT) enzymes, encoded by the
STE14A and STE14B (ICMT) genes, catalyze the methylation of
C-terminal isoprenylcysteines in Arabidopsis (Crowell et al.,
1998; Rodrıguez-Concepcion et al., 2000; Crowell and Kennedy,
2001; Chary et al., 2002). The products of these genes, ex-
pressed as recombinant proteins in Saccharomyces cerevisiae,
have been shown to methylate N-acetyl-S-trans,trans-farnesyl-
L-cysteine (AFC) and N-acetyl-S-all trans-geranylgeranyl-L-cys-
teine (AGGC), which are biologically relevant isoprenylcysteine
methyl acceptor substrates, but not N-acetyl-S-trans-geranyl-
L-cysteine (AGC), a biologically irrelevant isoprenylcysteine sub-
strate (Chary et al., 2002). Demethylation of isoprenylcysteine
methyl esters is catalyzed by isoprenylcysteine methylesterase
(ICME), which is encoded by the ICME gene (Deem et al., 2006).
The ICME gene encodes a 427–amino acid polypeptide with a
carboxyl esterase type B Ser active site and relatedness to sterol
esterases, juvenile hormone esterases, and carboxyl ester li-
pases. Furthermore, the ICME genewas shown, by expression of
recombinant protein in S. cerevisiae, to encode an isoprenylcys-
teine carboxyl methylesterase with high selectivity for biologi-
cally relevant isoprenylcysteinemethyl ester substrates (i.e., AFC
methyl ester and AGGC methyl ester, but not AGC methyl ester)
(Deem et al., 2006). Two additional genes exist in theArabidopsis
genome with sequence relatedness to ICME (At3g02410 and
At1g26120), but the protein products of these genes have not
been tested for ICME activity.
As described above, two geranylgeranylated proteins (ROP2
and ROP6) and at least one farnesylated protein negatively
Figure 2. ICMTox1 and ICMTox2 Plants Exhibit Increased Levels of STE14B mRNA and ICMT Activity.
(A) RT-PCR of STE14BmRNA from wild-type (Col-0), ICMTox1, and ICMTox2 lines is shown. The values at top indicate the amounts of input RNA added
to the RT-PCR. RT-PCR of 18S rRNA served as an internal loading control.
(B) The relative mRNA abundances for the lines shown in (A) were determined by image analysis using Image J software (National Institutes of Health).
The mRNA abundance of Col-0 is set to 1 and serves as the point of reference for ICMTox1 and ICMTox2. Error bars represent SE of three replicates.
Asterisks indicate significant differences from the control, as judged by Student’s t test (* P < 0.05).
(C) Quantitative assay of ICMT activity in membranes of Col-0, ICMTox1, and ICMTox2 lines. AFC and AGGC are biologically relevant isoprenylcysteine
methyl acceptor substrates. AGC is a biologically irrelevant negative control substrate. The TLC fluorogram shows product formation as a function of
time.
(D) Product formation was quantified by liquid scintillation of the excised TLC spots shown in (C). The results shown are representative of two
independent experiments.
Protein Methylation and ABA Signaling 3 of 15
regulate ABA signaling in Arabidopsis. However, it is not clear at
present where, or how, these isoprenylated proteins function in
ABA signaling. The observation that ggb mutations affect a
subset of ABA responses suggests that geranylgeranylated
negative regulators of ABA signaling more than likely regulate
events downstream of branch points in the ABA signaling path-
way or regulate some, but not all, ABA signaling pathways. The
stomata of ggb plants were found to exhibit an enhanced
response to ABA, consistent with the known role of ROP6 in
negative regulation of ABA-induced stomatal closure (Lemichez
et al., 2001; Johnson et al., 2005), but the response of ggb seeds
to ABA was normal, despite a report that ROP2 is involved in
negative regulation of ABA signaling in seeds (Li et al., 2001).
While this may seem like a contradiction, it is possible that PFT
activity in ggb plants is sufficient for the isoprenylation and
function of certain isoprenylated proteins, such as ROP2, but not
for others. Indeed, numerous reports exist of isoprenylated
proteins that are substrates of both PFT and PGGT I and others
that are strictly substrates of PFT or PGGT I (Trueblood et al.,
1993; Armstrong et al., 1995). Given this heterogeneity in the
specificity of PFT and PGGT I for certain CaaX proteins and the
existence of at least two geranylgeranylated and one farnesy-
lated protein involved in negative regulation of ABA signaling,
deconvoluting the complex roles of isoprenylcysteine methyla-
tion and demethylation in negative regulation of ABA signaling
poses a significant challenge. Nevertheless, to begin to address
this problem, and to clearly establish whether ICMT and ICME
expression influence ABA signaling, we analyzed Arabidopsis
plants that overexpress either ICMT or ICME. These analyses
demonstrated conclusively that ICMT is a negative regulator of
ABA signaling and ICME is a positive regulator of ABA signaling.
Furthermore, ABA was found to induce the expression of the
ICME gene, thereby increasing the ABA responsiveness of
Arabidopsis plants.
RESULTS
ICMT Is a Negative Regulator of ABA Signaling
Competitive inhibitors of ICMT, including AFC, cause an en-
hanced response to ABA in seed germination and stomatal
closure assays compared with solvent controls (Chary et al.,
2002). These results are consistentwith the hypothesis that ICMT
is a negative regulator of ABA signaling. However, the effects of
farnesylcysteine (FC) analog inhibitors of ICMT could be non-
specific. To definitively establish whether or not ICMT is a
negative regulator of ABA signaling, we generated transgenic
Arabidopsis plants that overexpress the STE14B (ICMT) gene
using a cauliflower mosaic virus (CaMV) 35S promoter to drive
transcription of the transgene. STE14B was chosen for this
purpose because it encodes an ICMT with lower apparent Km
values for biologically relevant isoprenylcysteine substrates and
dramatically higher specific activities than the STE14A-encoded
enzyme (Chary et al., 2002). As shown in Figure 2, two indepen-
dent ICMT-overproducing lines, called ICMTox1 and ICMTox2,
contained significantly higher levels of STE14BmRNA and ICMT
activity than control plants. As expected, the ICMT activity in
these plants efficiently methylated AFC and AGGC, but not AGC.
Interestingly, ICMTox1 and ICMTox2 plants exhibited a significant
reduction in ABA inhibition of seed germination (Figure 3). Even in
the absence of exogenous ABA, ICMTox1 and ICMTox2 seeds
germinated more rapidly than control seeds, suggesting
Figure 3. ICMT Overexpression Lines of Arabidopsis Exhibit Reduced
ABA Inhibition of Seed Germination Compared with Control Col-0 Plants.
Seed germination was scored for Col-0, ICMTox1, and ICMTox2 lines as a
function of time in the presence of 0, 0.5, 2.5, or 5.0 mM cis-ABA (each
data point represents three independent trials; 40 < n < 83). The SE is
indicated for all data points. Asterisks represent significant differences
from the Col-0 control of * P < 0.05, ** P < 0.01, and *** P < 0.001, as
determined by Student’s t test.
4 of 15 The Plant Cell
insensitivity to endogenous ABA. The ABA-insensitive pheno-
type of ICMTox1 and ICMTox2 seeds was apparent at all concen-
trations of ABA tested, up to and including 5.0 mM. The ICMTox1
and ICMTox2 lines also exhibited a significant decrease in ABA-
induced stomatal closure compared with control plants (Figure
4A). No significant differences were observed in the absence of
exogenous ABA, but statistically significant differences were
observed in the presence of exogenous ABA. These results dem-
onstrate that ICMT overexpression results in an ABA-insensitive
phenotype in several ABA response assays (i.e., inhibition of
seed germination and induction of stomatal closure). However,
given that ggbmutants are affected in a subset of ABA responses
(Johnson et al., 2005), we considered the possibility that ICMTox1
and ICMTox2 plants might also be affected in a subset of ABA
responses. To test this hypothesis, ICMTox1 and ICMTox2 plants
were tested for ABA inhibition of root growth and induction of
ABA-induced genes. As shown in Figure 4B, ABA inhibition of
root growth in ICMTox1 and ICMTox2 plants was identical to that in
control plants. However, as shown in Figure 5, ICMTox1 and
ICMTox2 seedlings exhibited reducedABA induction ofRD29A and
COR47 gene expression (Yamaguchi-Shinozaki and Shinozaki,
1993; Baker et al., 1994). The ABA induction of ABI5, RD22, and
COR15A, on the other hand, was not greatly altered in ICMTox
plants (Figure 5; data not shown for COR15A). Thus, like ggb
plants, ICMTox1 and ICMTox2 plants are affected in a subset
of ABA responses, suggesting that isoprenylated negative reg-
ulators of ABA signaling function downstream of at least one
branch point in the ABA signaling pathway or participate in some,
but not all, ABA signaling pathways. Together, these observa-
tions establish ICMT as a negative regulator of ABA signaling and
suggest that isoprenylated negative regulators of ABA signaling
are incompletely methylated in planta (i.e., overproduction of
ICMT would have no effect on ABA signaling if isoprenylated
negative regulators of ABA signaling were completely methyl-
ated in untransformed plants).
ICME Is a Positive Regulator of ABA Signaling
If ICMT is a negative regulator of ABA signaling, it follows that
ICME is a positive regulator of ABA signaling. To test this
hypothesis, we generated Arabidopsis plants that overexpress
the ICME gene, again using a CaMV 35S promoter to drive
transgene expression. As shown in Figure 6, two independent
ICME-overproducing lines, called ICMEox1 and ICMEox2, con-
tained significantly higher ICME mRNA levels and ICME activity
than control plants. In all of the independent transgenic lines
analyzed, none exhibited higher ICME expression than ICMEox1
and ICMEox2. This observation suggests that strong overexpres-
sion of the ICME gene is deleterious to the growth and develop-
ment of T1 transformants. Interestingly, the increase in ICME
activity in these lines (;50%) was less than the increase in ICME
mRNA abundance (;150%), suggesting the possibility of trans-
lational or posttranslational control of ICME expression. As ex-
pected, ICMEox1 and ICMEox2 lines exhibited a significantly
enhanced response to ABA inhibition of seed germination (Figure
7). Even in the absence of exogenous ABA, ICMEox1 and ICMEox2
seeds germinatedmore slowly than control seeds, suggesting an
enhanced response to endogenousABA. The ABA-hypersensitive
phenotype of ICMEox1 and ICMEox2 seeds was apparent at all
concentrations of ABA tested, up to and including 5.0 mM. The
ICMEox1 and ICMEox2 lines also exhibited a significant increase in
ABA-induced stomatal closure compared with control plants
(Figure 8A). In the absence of exogenous ABA, the stomatal
apertures of ICMEox1 and ICMEox2 plants were smaller than
those of control plants, suggesting hypersensitivity to endoge-
nous ABA. In the presence of exogenous ABA, a dramatic
overresponse was observed in the ICMEox lines, resulting in
stomata that were almost completely closed at 2.5 mM ABA.
These results demonstrate that ICME overexpression results in
an ABA-hypersensitive phenotype in multiple ABA response
assays (i.e., inhibition of seed germination and induction of
Figure 4. ICMT Overexpression Lines of Arabidopsis Exhibit Reduced ABA Induction of Stomatal Closure Compared with Control Col-0 Plants.
(A) Stomatal apertures were measured for Col-0, ICMTox1, and ICMTox2 lines after incubation of excised leaves for 2 h in the presence of 0, 0.5, or 2.5
mM cis-ABA. Stomatal apertures were measured from photomicrographs of epidermal peels and reported as averages of 50 stomata per data point.
(B) Root growth was measured after transferring seedlings (21 < n < 51) to plates containing various concentrations of ABA. The seedlings were placed
908 from vertical, and growth from the bend to the tip was recorded after 5 d.
The SE is shown for all data points. Asterisks represent significant differences from the control of * P < 0.05 and ** P < 0.001, as determined by Student’s
t test.
Protein Methylation and ABA Signaling 5 of 15
Figure 5. ICMT Overexpression Lines of Arabidopsis Exhibit Reduced ABA Induction of RD29A and COR47 Gene Expression Compared with Control
Col-0 Plants.
Total RNA fromCol-0, ICMTox1, and ICMTox2 seedlings was analyzed by RT-PCR for the presence of RD29A,COR47,RD22, ABI5, and Actin8mRNA (as
a loading control). The bands shown were photographed from the same gel but were not side by side. The SE of two replicates is indicated for all data
points. Asterisks represent significant differences from the corresponding Col-0 control of * P < 0.05, ** P < 0.01, and *** P < 0.001, as determined by
Student’s t test.
6 of 15 The Plant Cell
stomatal closure). However, as with the ICMTox1 and ICMTox2
lines, ICMEox1 and ICMEox2 did not show a clear change in ABA
inhibition of root growth (ICMEox1 and ICMEox2 exhibited statis-
tically significant differences in ABA inhibition of root growth
comparedwith ecotype Columbia [Col-0] at 0.5 and 2.5mMABA,
respectively, but not at other ABA concentrations; Figure 8B). As
shown in Figure 9, the expression of ABA-induced geneswas not
higher in ICMEox1 and ICMEox2 seedlings than in Col-0 seedlings.
Given that the ICMTox1 and ICMTox2 lines exhibited reduced ABA
induction of RD29A and COR47 gene expression, it was some-
what surprising that expression of these genes was not higher in
the presence of ABA in ICMEox1 and ICMEox2. This result either
means that the modest overexpression of ICME in ICMEox1 and
Figure 6. ICMEox1 and ICMEox2 Plants Exhibit Increased Levels of ICME
mRNA and ICME Activity.
(A) RT-PCR of ICME mRNA from wild-type (Col-0), ICMEox1, and
ICMEox2 lines is shown. The values at top indicate the amounts of input
RNA added to the RT-PCR. RT-PCR of Actin8mRNA served as a loading
control.
(B) The relative mRNA abundances for the lines shown in (A) were
determined by image analysis using Image J software. The mRNA
abundance of Col-0 is set to 1 and serves as the point of reference for
ICMEox1 and ICMEox2.
(C) Quantitative assay of ICME activity in membranes of Col-0, ICMEox1,
and ICMEox2 lines. The activity of Col-0 is set to 1 and serves as the point
of reference for ICMEox1 and ICMEox2.
Error bars represent SE of five replicates. Asterisks indicate significant
differences from the control of * P < 0.05, ** P < 0.01, and *** P < 0.001, as
judged by Student’s t test.
Figure 7. ICME Overexpression Lines of Arabidopsis Exhibit Enhanced
ABA Inhibition of Seed Germination Compared with Control Col-0 Plants.
Seed germination was scored for Col-0, ICMEox1, and ICMEox2 lines as a
function of time in the presence of 0, 0.5, 2.5, or 5.0 mM cis-ABA (each
data point represents four independent trials; 91 < n < 138). The SE is
indicated for all data points. Asterisks represent significant differences
from the Col-0 control of * P < 0.05, ** P < 0.01, and *** P < 0.001, as
determined by Student’s t test.
Protein Methylation and ABA Signaling 7 of 15
ICMEox2 is insufficient to affect this particular ABA response or
other factors limit the ABA induction of RD29A and COR47 gene
expression. Thus, like the ICMTox1 and ICMTox2 lines, ICMEox1
and ICMEox2 are affected in a subset of ABA responses. Taken
together, these observations support the hypothesis that ICME is
a positive regulator of ABA signaling, at least with respect to ABA
inhibition of seed germination and ABA-induced stomatal clo-
sure.
ABA Induces ICME Gene Expression
The results described above firmly establish that ICMT and ICME
regulate ABA responsiveness in Arabidopsis. To determine
whether ABA affects the expression of the ICMT and/or ICME
genes, seedlings were grown for 4 d on half-strength Murashige
and Skoog (MS) plates containing 1% sucrose and 0.8% agar
and then transferred to plates containing 0.5 or 5.0 mM cis-ABA,
0.1mMnaphthalene acetic acid (NAA), or an equivalent volume of
DMSO for 24 h. These concentrations were chosen because they
induce responses in standard physiological assays (e.g., inhibi-
tion of seed germination and induction of stomatal closure for
ABA and initiation of lateral root formation for NAA; Johnson
et al., 2005). Interestingly, 5.0 mMABA caused an approximately
threefold accumulation of ICMEmRNA (Figure 10) and a twofold
increase in ICME activity in wild-type Col-0 seedlings (Figure 11).
As observed in Figure 6, the increase in ICMEmRNA abundance
was greater than the increase in ICME activity, suggesting
translational or posttranslational control of ICME gene expres-
sion. These results are consistent with the hypothesis that ABA
promotes ABA responsiveness of plant cells via induction of
ICME gene expression and demethylation (i.e., inactivation) of
isoprenylated negative regulators of ABA signaling. Having
established that ABA induces ICME gene expression in wild-
type seedlings, we investigated whether ABA induction of ICME
expression was affected in ICMTox and ICMEox seedlings. As
shown in Figure 11, ABA induction of ICME expression was
decreased in ICMTox1 and ICMTox2 seedlings, confirming the
ABA-insensitive phenotype of these plants. Not surprisingly,
ABA induction of ICME expression was also decreased in
ICMEox seedlings, presumably because of the elevated ICME
expression in these plants. By contrast, no regulation of STE14A
or STE14B expression by ABA or NAA was observed (data not
shown). These results are supported by publicly available micro-
array data, which reveal a fourfold to fivefold increase in ICME
mRNA abundance following ABA treatment (microarray data
visualized using the AtGenExpress Visualization Tool at http://
jsp.weigelworld.org/expviz/expviz.jsp). Microarray data also
suggest roles for ICME in leaf senescence, seed maturation,
abiotic stress responses (i.e., osmotic, salt, UV-B light, and heat
stress), and defense responses to bacterial and fungal patho-
gens (see Supplemental Data Set 1 online). Consistent with our
findings, microarray data do not support the hypothesis that
expression of STE14A or STE14B is ABA- or auxin-responsive.
DISCUSSION
In this article, ICMT overexpression is shown to cause an ABA-
insensitive phenotype in seed germination and stomatal closure
assays and reduced ABA induction ofRD29A,COR47, and ICME
gene expression (Figures 3, 4, 5, and 11). However, ICMT
overexpression had no effect on ABA inhibition of root elonga-
tion. These observations are consistent with the hypothesis that
ICMT is a negative regulator of ABA signaling and, as suggested
by the phenotype of ggb mutants, either functions downstream
of at least one branch point in the ABA signaling pathway or
functions in a subset of ABA signaling pathways (Bonetta and
McCourt, 1998). These data also suggest that isoprenylated
negative regulators of ABA signaling are not completely meth-
ylated in wild-type Arabidopsis plants, because overexpression
Figure 8. ICME Overexpression Lines of Arabidopsis Exhibit Enhanced ABA Induction of Stomatal Closure Compared with Control Col-0 Plants.
(A) Stomatal apertures were measured for Col-0, ICMEox1, and ICMEox2 lines after incubation of excised leaves for 2 h in the presence of 0, 0.5, or 2.5
mM cis-ABA. Stomatal apertures were measured from photomicrographs of epidermal peels and reported as averages of 50 stomata per data point.
(B) Root growth was measured after transferring seedlings (22 < n < 32) to plates containing various concentrations of ABA. The seedlings were placed
908 from vertical, and growth from the bend to the tip was recorded after 5 d.
The SE is shown for all data points. Asterisks represent significant differences from the control of * P < 0.01 and ** P < 0.001, as determined by Student’s
t test.
8 of 15 The Plant Cell
Figure 9. ICME Overexpression Lines of Arabidopsis Do Not Exhibit Dramatically Altered ABA Induction of Gene Expression Compared with Control
Col-0 Plants.
Total RNA from Col-0, ICMEox1, and ICMEox2 seedlings was analyzed by RT-PCR for the presence of RD29A, COR47, RD22, ABI5, and Actin8 mRNA
(as a loading control). The bands shown were photographed from the same gel but were not side by side. The SE of two replicates is indicated for all data
points. Asterisks represent significant differences from the corresponding Col-0 control of * P < 0.05, ** P < 0.01, and *** P < 0.001, as determined by
Student’s t test.
Protein Methylation and ABA Signaling 9 of 15
of ICMT would not be expected to have an effect if they were.
Consistent with this suggestion, immunoblots using a pan-ROP
antibody demonstrated that ICMT overexpression increased the
membrane association of ROP proteins, although the antiserum
used did not allow for the identification of which ROP proteins
were affected (see Supplemental Figure 1 online). Of the 11
knownArabidopsisROPproteins, 8 are known or predicted to be
isoprenylated, whereas 3 (i.e., the type II ROPs) are membrane-
associated via an isoprenylation-independent mechanism (Lavy
et al., 2002; Lavy and Yalovsky, 2006). ROP10, which has been
implicated in negative regulation of ABA signaling (Zheng et al.,
2002; Xin et al., 2005), is a type II ROP. However, ROP6, which
has a CSIL motif at its C terminus with an upstream polybasic
domain (Lemichez et al., 2001), and ROP2, which possesses a
C-terminal CAFL motif and can be geranylgeranylated in vitro
(see Supplemental Figure 2 online), are also negative regulators
of ABA signaling (Li et al., 2001). Thus, more than one isopreny-
lated negative regulator of ABA signaling has been identified.
These ROPs, however, are geranylgeranylated and thus do not
explain the enhanced response to ABA of era1 mutants, which
lack the b-subunit of PFT. Farnesylated negative regulators of
ABA signaling have not, to date, been identified.
The data in Figures 7 and 8 demonstrate that overexpression
of the ICME gene causes an enhanced response to ABA in seed
germination and stomatal closure assays. It is interesting that
ICME overexpression caused a slight, but significant, decrease
in stomatal apertures even in the absence of exogenous ABA
(Figure 8), suggesting an enhanced response to endogenous
ABA. That ABA-induced gene expression in ICMEox1 and
ICMEox2 did not exceed that in Col-0 attests to the modest
overexpression of ICME in these lines or suggests the presence
of other factors that limit ABA-induced gene expression. How-
ever, a close examination of the data in Figure 9 reveals sig-
nificant differences in ABA-induced gene expression between
Col-0 and ICMEox lines for COR47 and ABI5. Both genes were
expressed at significantly lower levels in the absence of exog-
enous ABA and were induced to a greater extent in the presence
of exogenous ABA in ICMEox1 and ICMEox2 seedlings. Conse-
quently, at 5.0 mM ABA, no significant differences in COR47
or ABI5 gene expression were observed between Col-0 and
ICMEox lines. These data can be interpreted, albeit with caution,
to mean that increased ABA induction of COR47 and ABI5 gene
expressionwas observed in ICMEox1 and ICMEox2. Overall, these
data demonstrate that ICME is a positive regulator of ABA
signaling and, like ICMT, affects some, but not all, ABA re-
sponses. In total, the data in Figures 3, 4, 5, 7, and 8 support the
hypothesis that isoprenylcysteine methylation is required for the
function of isoprenylated negative regulators of ABA signaling
and isoprenylcysteine demethylation attenuates the function of
these proteins.
Until recently, no T-DNA insertions in either STE14A or
STE14B were available. However, a T-DNA insertion three nu-
cleotides upstream of the TGA codon of STE14A was recently
reported (Bracha-Drori et al., 2008). This mutant exhibited no
detectable STE14A mRNA and no observable phenotype, sug-
gesting that its function is either dispensable or redundant with
that of STE14B. Transgenic plants containing an STE14B RNA
interference (RNAi) construct did not exhibit a significant de-
crease in STE14B mRNA abundance, but a subtle phenotype
was observed (Bracha-Drori et al., 2008), suggesting that either
STE14B silencing occurred at the level of translation or another
gene was affected by the RNAi construct (no data were pre-
sented to demonstrate reduced ICMT protein or activity). These
plants exhibited altered phylotaxis, multiple axillary buds, and
fasciated stems, but they did not exhibit an ABA-hypersensitive
phenotype (Bracha-Drori et al., 2008). Given the ABA hypersen-
sitivity of era1, plp, ggb, and ICMEox plants, this observation is
surprising, but it is impossible to interpret without evidence of
reduced ICMT activity in STE14B RNAi plants.
The SALK collection includes a mutant with a T-DNA insertion
in the ICME gene, but the insertion does not cause a knockout
phenotype and, therefore, was not analyzed in detail or included
in the body of this report. Nevertheless, the SALK_010304 line,
which has a T-DNA insertion in intron 1 of the ICME gene,
exhibited decreased ICME mRNA abundance and an ABA-
insensitive phenotype in seed germination assays (see
Figure 10. ABA Induces ICME Gene Expression.
(A) RT-PCR of ICMEmRNA from Arabidopsis seedlings (Col-0) grown for
4 d and then exposed to 5.0 mM cis-ABA, 0.1 mM NAA, or an equivalent
volume of DMSO for 24 h. The values at top indicate the amounts of input
RNA added to the RT-PCR. RT-PCR of Actin8mRNA served as a loading
control.
(B) The relative mRNA abundances for the treatments shown in (A) were
determined by image analysis using Image J software. Error bars
represent SE. Asterisks indicate a significant difference from the control,
as judged by Student’s t test (** P < 0.01). The results shown are
representative of three independent experiments.
10 of 15 The Plant Cell
Supplemental Figure 3 online). These results support the con-
clusion that ICME is a positive regulator of ABA signaling.
With known and unknown isoprenylated proteins affecting
ABA signaling, it is not possible at this time to link isoprenylcys-
teinemethylation and demethylation of a specific protein or set of
proteins to the changes in ABA responsiveness observed in
ICMTox and ICMEox plants. It is unlikely that the effects of ICMT or
ICME overexpression are due to changes in themethylation state
of free isoprenylcysteines (i.e., in lieu of isoprenylated proteins),
because free isoprenylcysteines are present in wild-type Arabi-
dopsis plants at virtually undetectable levels (data not shown).
However, plants with T-DNA insertions in the FCLY gene, which
encodes a specific FC lyase (Figure 1), accumulate FC and
exhibit an enhanced response to ABA in seed germination and
stomatal closure assays (Crowell et al., 2007). The ABA hyper-
sensitivity of fcly plants, which establishes the FCLY gene as
another negative regulator of ABA signaling, is presumably the
result of FC inhibition of ICMT (Figure 1) (Chary et al., 2002). Even
if the effects described here are the result of changes in the
methylation state of free isoprenylcysteines, the conclusions that
ICMT is a negative regulator, and ICME a positive regulator, of
ABA signaling are nonetheless valid.
Surprisingly, like ggb plants, ICMTox and ICMEox plants
exhibited no obvious developmental phenotype. Because era1-2
and plp plants exhibit enlarged meristems and supernumerary
floral organs, this phenotype might be expected in ICMEox
plants. That no such phenotype was observed suggests that
the isoprenylated protein(s) involved in meristem development is
(1) fullymethylated, even in the presence of ICMEoverproduction
(i.e., because of high ICMT activity in meristems), or (2) unaf-
fected by isoprenylcysteine methylation/demethylation. Alterna-
tively, it is also possible that the modest overproduction of ICME
in ICMEox plants is insufficient to affect the function of this protein
(s). It is noteworthy that clv3-7 mutants, which have enlarged
meristems and increased floral organ numbers, exhibit de-
creased STE14A mRNA accumulation (see Supplemental Data
Figure 11. Analysis of ABA Induction of ICME mRNA Accumulation and ICME Activity in Col-0, ICMTox, and ICMEox Seedlings Treated with 0.5 or 5.0
mM cis-ABA or an Equivalent Volume of DMSO.
(A) RT-PCR of ICME mRNA from Col-0, ICMTox, and ICMEox lines treated with 0, 0.5, or 5.0 mM cis-ABA is shown.
(B) Relative ICME activity in membranes of Col-0, ICMTox, and ICMEox lines. The ICME activity of the solvent control for each line is set to 1 and serves
as the point of reference for the corresponding 0.5 and 5.0 mMABA samples. Error bars represent SE. Asterisks indicate significant differences from the
0 mM ABA control of * P < 0.05, ** P < 0.01, and *** P < 0.001, as judged by Student’s t test. The results shown are representative of three independent
experiments.
Protein Methylation and ABA Signaling 11 of 15
Set 1 online), suggesting a potential role for the STE14A-
encoded ICMT in meristem development.
The data shown in Figures 10 and 11 demonstrate that ABA
causes the accumulation of ICMEmRNA, whereas NAA does not
significantly affect ICME mRNA abundance. This result is sup-
ported by publicly available microarray data, which reveal a
fourfold to fivefold increase in ICMEmRNA abundance following
ABA treatment (microarray data visualized using the AtGenEx-
press Visualization Tool at http://jsp.weigelworld.org/expviz/
expviz.jsp). By contrast, no change in STE14A or STE14B
mRNA abundance in the presence of exogenous ABA or NAA
was observed (data not shown).Whether ABA regulation of ICME
expression is due to increased ICME transcription, decreased
ICME mRNA degradation, or both is not known, but the obser-
vation that ICME is upregulated in response to ABA supports the
hypothesis that ABApromotes ABA responsiveness of plant cells
via increased ICME expression and subsequent demethylation
(i.e., loss of function) of isoprenylated negative regulators of ABA
signaling. This positive feedback loop, shown in Figure 12,
ensures that ABA signaling is not transient, a property that
makes biological sense given the role of ABA in long-term
responses, such as seed dormancy and stomatal closure under
prolonged drought conditions. This finding is also consistent with
previous reports of ABA-induced positive feedback regulation of
ABA biosynthesis (Xiong et al., 2002; Barrero et al., 2006). Other
microarray data indicate that ICME expression is initially down-
regulated by osmotic stress, salt stress, UV-B light stress, and
heat stress but is strongly upregulated after several hours by
these abiotic stresses (see Supplemental Data Set 1 online). This
is particularly interesting in view of the role of ABA in abiotic
stress responses. Future experiments will address the mecha-
nism of regulation of ICME gene expression by ABA and envi-
ronmental stimuli. Ultimately, the identification of all factors that
regulate ICME and/or ICMT expression will be necessary for a
complete understanding of how the balance between isoprenyl-
cysteine methylation and demethylation affects ABA signaling
and other physiological processes in plants.
Drought is a major cause of variability in crop yields from year
to year. The data shown here and elsewhere (Crowell et al., 2007)
demonstrate that ICMT and FCLY are negative regulators, and
ICME is a positive regulator, of ABA signaling. Thus, under-
expression of ICMT or FCLY, or overexpression of ICME, would
be expected to cause an enhanced response to ABA in trans-
genic plants. Using these strategies, singly or in combination,
and controlling transgene expression with a drought-inducible,
guard cell–specific promoter might result in plants that are
resistant to drought, and possibly salt stress, without deleterious
effects on germination or growth. This work has the potential to
stabilize crop production, even in years of severe drought.
METHODS
Plant Growth Conditions
Arabidopsis thaliana seeds were surface-sterilized according to the
following procedure: 95% ethanol for 5 min, 20 to 50% bleach for 5 to
10 min, and five washes in sterile deionized water. Seeds were then
suspended in sterile 0.1% agar, imbibed on half-strength MS plates
containing 1% sucrose and 0.8% agar, treated for 3 d at 48C, and
germinated in a vertical orientation at 228C under long-day conditions (18
h of white light at 100 mmol·m22·s21 followed by 6 h of dark). Seedlings
were harvested after 4 d of growth for extraction of RNA or microsomal
membranes or transferred to ProMix and grown under the same condi-
tions for propagation or analysis of stomatal function. Plants were
irrigated and fertilized from below with a standard mixture of macronu-
trients and micronutrients.
Plant Transformation
To generate the ICMTox1 and ICMTox2 lines, the Col-0 ecotype of
Arabidopsis was transformed with a pCL108 construct harboring the
CaMV 35S promoter and the STE14B coding sequence. pCL108 was
derived fromplasmid YY217 (Yamamoto et al., 2001) by replacing the sm-
RSGFP fragment of YY217 with an XbaI–KpnI fragment containing the
STE14B coding sequence. To generate the ICMEox1 and ICMEox2 lines,
the ICME coding sequence was amplified by RT-PCR, confirmed by DNA
sequence analysis, and inserted into the EcoRI and HindIII sites of the
vector pH2GW7 (Karimi et al., 2002), downstream of the CaMV 35S
promoter. Agrobacterium tumefaciens strain GV3101 (pMP90) (Koncz
and Schell, 1986) and the floral dip method (Clough and Bent, 1998) were
used for plant transformation. T1 transformants were selected on half-
strength MS plates containing 1% sucrose, 0.8% agar, and 200 mg/mL
gentamycin (ICMTox1 and ICMTox2) or 50 mg/mL hygromycin (ICMEox1
and ICMEox2). Transgenic plant lines were propagated to the T3 gener-
ation and scored for homozygosity by PCR and nonsegregation of the
antibiotic resistance marker.
Figure 12. Model for Positive Feedback Regulation of ABA Signaling via
Induction of ICME Expression and Demethylation (i.e., Inactivation) of
Isoprenylated Negative Regulators of ABA Signaling.
ABA promotes seed dormancy and stomatal closure but also increases
the expression of the ICME gene, a positive regulator of ABA signaling.
To date, the only isoprenylated negative regulators of ABA signaling to be
identified are ROP2 and ROP6 (Lemichez et al., 2001; Li et al., 2001).
Consequently, the isoprenylated protein shown is modified by a gera-
nylgeranyl moiety and represents either ROP2 or ROP6. However, at
least one unidentified farnesylated negative regulator of ABA signaling
also exists in Arabidopsis. A hypothetical integral membrane isoprenyl-
protein receptor is shown.
12 of 15 The Plant Cell
RT-PCR Analysis of ICMTox and ICMEox Lines and ABA Induction
of ICME
Total RNA was isolated from seedlings of ICMTox1, ICMTox2, ICMEox1,
ICMEox2, and wild-type Col-0 plants using TRIzol reagent according to
the manufacturer’s instructions (Invitrogen/Life Technologies). RT-PCR
was then performed to analyze ICMT or ICME transcript levels using
different amounts of input RNA (250 to 2 ng), 5 pmol of forward primer, 5
pmol of reverse primer, 18S rRNA competimer/primer pairs (Ambion) or
Actin8 primers as a loading control, and the Platinum Quantitative RT-
PCR Thermoscript One-Step system (Invitrogen/Life Technologies) in a
total reaction volume of 25 mL. The STE14B forward and reverse primers
were ICMT-59 (59-GTGACACCGGGTTCAGACAGTTAA-39) and ICMT-39
(59-CAGTTTACAAAAGGAACACCAGAA-39), and the ICME forward and
reverse primerswere ICME-59 (59-GGTAGGCTATCGATGGATGACAA-39)
and ICME-39 (59-AGGACTCTTCTCCTTCCATTATG-39). RT-PCR condi-
tions included a 20-min reverse transcription step at 508C, followed by a
5-min presoak at 958C and 25 to 35 cycles of the following PCR program:
958C for 30 s; 538C for 30 s; and 728C for 90 s. A postsoak was performed
at 728C for 7 min to ensure complete product synthesis. RT-PCR
products were resolved by agarose gel electrophoresis and visualized
by ethidium bromide staining.
ICMT Assays
For ICMT assays,Arabidopsis extractswere prepared by grinding 4-d-old
seedlings with a mortar and pestle at 48C in a homogenization buffer
containing 50 mMHEPES (pH 7.4), 500 mMmannitol, 5 mM EDTA, 5 mM
DTT, and Complete protease inhibitors (Roche Diagnostics). Extracts
were filtered through four layers of cheesecloth and centrifuged at 8000g
for 10 min at 48C. Membranes were then sedimented from Arabidopsis
extracts at 100,000g for 60min at 48Cand resuspended in a resuspension
buffer containing 2.5 mM HEPES (pH 7.0), 250 mM mannitol, and 1 mM
DTT. Membrane aliquots were stored at 2808C in the presence of 15%
glycerol. ICMT assays were performed essentially as described (Hrycyna
and Clarke, 1990; Crowell et al., 1998) in the presence of 60 mg of
membrane protein, S-adenosyl-L-[3H-methyl]methionine (GE Healthcare
Life Sciences) as a methyl donor (24 mM, 2.5 Ci/mmol, 60 mCi/mL), 200
mM AFC or AGGC as a methyl acceptor (200 mM AGC was used as a
negative control), and 100mMHEPES (pH 7.0), in a total volume of 50mL.
Stock solutions of AFC, AGGC, and AGC (BioMol) were prepared in
DMSOat a concentration of 10mM.Reactionswere incubated at 308C for
1 h, terminated by the addition of 50 mL of 90% (v/v) methylene chloride,
9.75% (v/v) methanol, and 0.25% (v/v) acetic acid, mixed vigorously, and
centrifuged for 1 min in a microcentrifuge. A 10-mL portion of the organic
phase was analyzed by thin layer chromatography (TLC) using a Poly-
gram Sil G plastic-backed silica gel plate (Aldrich) and 90% methylene
chloride, 9.75% methanol, and 0.25% acetic acid as a mobile phase.
Plates were sprayed with En3hance fluorographic reagent (NEN/DuPont),
and fluorography was performed for 1 to 3 d at 2808C with X-Omat AR
film (Eastman Kodak). ICMT activity was quantified by liquid scintillation
of excised TLC spots.
ICME Assays
To prepare isoprenylcysteine methyl ester substrates, recombinant
Arabidopsis ICMT was expressed in Saccharomyces cerevisiae strain
SM1188 (MATa ste14::TRP1, leu2, ura3, trp1, his4, can1) and used to
generate the methyl ester of AFC. STE14B expression was induced by
shifting log-phase cells grown at 308C in CSM-uramedium containing 2%
glucose into CSM-ura medium containing 2% galactose. After 14 h at
308C in galactose medium, the cells were sedimented and lysed in lysis
buffer containing 100 mM Tris-HCl (pH 7.5), 1 mM DTT, 20% (v/v)
glycerol, andComplete protease inhibitors by vortexing in the presence of
acid-washed glass beads. Yeast extracts were centrifuged at 8000g for
10 min at 48C, and membranes were prepared from the supernatants by
ultracentrifugation at 100,000g for 60 min at 48C. ICMT reactions were
performed as described above except that En3hance fluorographic
reagent was not used. Plates were exposed directly to Kodak XAR-5
film (Eastman Kodak) for 3 to 5 d at 2808C, after which TLC spots
corresponding to AFCmethyl ester were excised, eluted into ethanol, and
stored at 2808C at a radiochemical concentration of 10,000 cpm/mL.
ICME assayswere performed essentially as described (Deem et al., 2006)
in the presence of 80mMHEPES (pH 7.5), 5mMMgCl2, 100mMNaCl, up
to 100 mg of membrane protein from 4-d-old Arabidopsis seedlings
prepared as described above, and 30,000 cpm of AFC methyl ester
substrate (2.5 Ci/mmol, 0.27 mCi/mL) in a reaction volume of 50 mL.
Reactions were incubated at 308C for various times in sealed vials, and
volatile [3H]methanol was captured into scintillation cocktail.
Seed Germination Assays
Arabidopsis seeds were harvested from control and experimental plants,
which were grown together under identical conditions. Seeds were
surface-sterilized as described above, suspended in sterile 0.1% agar,
and placed on half-strength MS plates containing 1% sucrose and 0.8%
agar in the dark at 228C as described (Cutler et al., 1996). Seeds from
control and experimental plants were sown in rows on the same plates
under identical conditions, and germination (radical emergence) was
scored at various times in the presence of various concentrations of
exogenous ABA with a dissecting microscope.
Stomatal Closure Assays
Mature rosette leaves of Arabidopsis were excised and incubated for 2 h
in the presence of various concentrations of ABA or an equivalent volume
of DMSO (as a solvent control) in 10mL of water. Abaxial epidermal peels
were then prepared by peeling away the leaf surface with Scotch tape.
Epidermal peels were stained with toluidine blue, mounted on a micro-
scope slide, and visualized with a Nikon Eclipse E600 microscope in-
terfaced to a SPOT digital camera. Data are recorded as average widths
of individual apertures (n = 50 per sample). In random order, D.N.C.
excised and incubated leaves in the presence of various concentrations
of ABA. D.H.H. prepared epidermal peels and photographed and mea-
sured stomatal apertures without knowledge of sample identities.
Root Growth Assays
Arabidopsis seeds were surface-sterilized as described above, suspen-
ded in sterile 0.1% agar, imbibed on half-strength MS plates containing
1% sucrose and 0.8% agar, treated for 3 d at 48C, and germinated in a
vertical orientation at 228C. After 2 d, seedlings were transferred to plates
containing various concentrations of ABA, turned 908, and visualized after
an additional 5 d of growth. Root length from the bend to the tip was
measured in order to rule out differences in germination time.
Analysis of ABA-Induced Gene Expression
Arabidopsis seedlings were germinated as described above in a vertical
orientation at 228C under long-day conditions (18 h of white light at 100
mmol·m22·s21 followed by 6 h of dark), grown for 6 d, and then transferred
to plates containing ABA (0.5, 2.5, or 5.0 mM) or an equivalent volume of
DMSO for 24 h. RNAwas extracted using TRIzol reagent according to the
manufacturer’s instructions, and RT-PCR was performed to determine
the relative accumulation of mRNA corresponding to the ABA-induced
genes RD29A, COR47, RD22, ABI5, and COR15A. RT-PCR was per-
formed as described above with the following primers: RD29A-59
Protein Methylation and ABA Signaling 13 of 15
(59-TACCGGAGATTGCTGAGTCTTT-39), RD29A-39 (59-CTCTGTTTTG-
GCTCCTCTGTTT-39), COR47-59 (59-AGGTTACGGATCGTGGATTG-39),
COR47-39 (59-CTTCCTCTTCAGTGGTCTTGG-39), RD22-59 (59-CCAAC-
GTCCAAGTAGGTAAAGG-39), RD22-39 (59-GCGAATGGGTACTTCTGT-
TTGT-39), ABI5-59 (59-ATTGACCCTTGACGAGTTCC-39), ABI5-39 (59-CAA-
CCATTCCCATTTGCTGT-39), COR15A-59 (59-ATGGCTTCTTCTTTCCAC-
AGCG-39), COR15A-39 (59-CTTTGTGGCATCCTTAGCCTCT-39), ACT8-59
(59-ATTGCCTCGTCGTCTTCAGCTTC-39), ACT8-39 (59-GTACGACCACT-
GGCGTAAAGTGA-39).
Statistical Methods
Throughout this report, data are represented as means 6 SE. Unlike the
SD, the SE takes into account sample size and is, thus, a more meaningful
measure of error. In all experiments, ICMTox and ICMEox plants are
compared with the Col-0 control at the same time and/or ABA concen-
tration. Because these are pair-wise comparisons, a Student’s t test was
performed to assess the statistical significance of all differences, and only
differences with P < 0.05 (95% confidence) were considered significant.
Accession Numbers
Sequence data from this article can be found in the Arabidopsis Genome
Initiative database under the following accession numbers: STE14A,
At5g23320 (The Arabidopsis Information Resource [TAIR] accession
locus 505006585); STE14B (ICMT), At5g08335 (TAIR accession locus
2166963); ICME, At5g15860 (TAIR accession locus 2143236); PLP,
At3g59380; ERA1, At5g40280; GGB, At2g39550; STE24, At4g01320;
FACE-2, At2g36305.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. The Effect of ICMT Overexpression on ROP
Membrane Association in Arabidopsis Seedlings.
Supplemental Figure 2. ROP2 Geranylgeranylation in Vitro.
Supplemental Figure 3. Analysis of an Arabidopsis icme-1 Mutant.
Supplemental Data Set 1. Microarry Data for ICME, STE14A, and
STE14B (ICMT).
ACKNOWLEDGMENTS
This work was supported by USDA Grant 2003-02151 and National
Science Foundation Grant MCB-0642957 to D.N.C.
Received June 4, 2007; revised October 4, 2008; accepted October 11,
2008; published October 28, 2008.
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Protein Methylation and ABA Signaling 15 of 15
DOI 10.1105/tpc.107.053389; originally published online October 28, 2008;Plant Cell
David H. Huizinga, Olutope Omosegbon, Bilal Omery and Dring N. Crowell ArabidopsisIsoprenylcysteine Methylation and Demethylation Regulate Abscisic Acid Signaling in
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