isoprenylcysteine methylation and demethylation regulate ...isoprenylcysteine methylation and...

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.


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.


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.


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.


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.


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.


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.


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.


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.


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


(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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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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