Gene 326 (2004) 35 – 41

www.elsevier.com/locate/gene

Genomic organization and regulation of the LeIMP-1 and LeIMP-2

genes encoding myo-inositol monophosphatase in tomato
Jean C. Styer 1, James Keddie 2, Jeremiah Spence, Glenda E. Gillaspy *
Virginia Tech, Department of Biochemistry and Fralin Biotechnology Center, 306, Blacksburg, VA 24061, USA
Received 10 February 2003; received in revised form 20 August 2003; accepted 20 September 2003

Received by W. Martin

Abstract

Myo-inositol (inositol) monophosphatase (IMP), an enzyme which catalyzes the synthesis of free inositol from various inositol
monophosphates, is encoded by a small multigene family in many organisms. The tomato IMP gene family encodes three IMP isoforms with
identical in vitro biochemical properties. To determine the role of each tomato LeIMP gene in plant growth, we isolated the genomic DNA
copies of the LeIMP-1 and LeIMP-2 genes. The LeIMP-1 gene spans approximately 5.8 kb and consists of 12 exons, whereas the LeIMP-2
gene consists of an uninterrupted, single open reading frame (ORF). We have previously shown that steady-state levels of LeIMP-2 mRNA
were very low in comparison to LeIMP-1 and LeIMP-3 mRNA levels. To determine whether LeIMP-2 gene expression was spatially
restricted to a discreet domain within the plant we constructed transgenic plants containing an LeIMP-2 promoter::uidA gene fusion. Analysis
of transgenic seedlings revealed that the LeIMP-2 promoter directed gene expression within epidermal and cortex cells of specific stem/leaf
junctions in an abaxial-specific pattern and in the shoot apical meristem. Further, inositol, the product of IMP catalysis, and Li+, an inhibitor
of IMP catalysis, decreased expression of the LeIMP-2 promoter as measured by a decrease in h-glucuronidase activity after treatment.
D 2003 Elsevier B.V. All rights reserved.

Keywords: Transgenic plant; h-Glucuronidase; Intronless

1. Introduction pounds ononitol and pinitol, and inositol hexakisphosphate

Myo-inositol (inositol) is required for normal growth of
plants, animals, yeast and some microorganisms (Loewus
and Murthy, 2000). In yeast and animal cells, inositol is
primarily incorporated into phosphatidylinositol phosphates
(PIPs) that function as precursors for the inositol trisphos-
phate (IP3) signaling pathway (Mitchell, 1986). In plants,
however, inositol is used for PIP synthesis as well as for the
synthesis of many other compounds such as indole acetic
acid (IAA) conjugates, galactinol, the stress-related com-
Abbreviations: Inositol, myo-inositol; GUS, h-glucuronidase; bp,
basepair(s); kb, kilobase(s); ORF, open reading frame; UTR, untranslated
region; IMP, inositol monophosphatase.
* Corresponding author. Tel.: +540-231-1850; fax: +540-231-7126.
E-mail address: gillaspy@vt.edu (G.E. Gillaspy).
1
Current address: Monsanto Company, 700 Chesterfield Village
Parkway North, St. Louis, MO 63198.
2
Current address: Mendel Biotechnology, 21375 Cabot Boulevard,
Hayward, CA 94545.
(IP6) (Loewus, 1990).
The first step in inositol synthesis is the conversion of
glucose-6-phosphate to inositol 1-phosphate (IP) (Loewus
and Murthy, 2000; Majumder et al., 1997). The last step of
synthesis, the dephosphorylation of IP, is catalyzed by the
enzyme myo-inositol monophosphatase (IMP; EC 3.1.3.25)
(Parthasarathy et al., 1994). IMP is also required to regen-
erate inositol from IP3 (Berridge et al., 1989). Two genes
encoding IMP are present in humans (McAllister et al.,
1992; Yoshikawa et al., 2000), mice (Shamir et al., 2001)
and yeast. There are three genes encoding IMP in tomato
(Gillaspy et al., 1995). The three tomato IMP cDNAs
(LeIMPs) encode distinct, but highly conserved IMP
enzymes that are catalytically active in vitro. All three
enzymes are inhibited by millimolar concentrations of LiCl.
The expression of these three LeIMP genes is developmen-
tally regulated, as is the accumulation of LeIMP proteins.
Maximal levels of IMP are observed in plant tissues
undergoing rapid cell divisions, such as light-grown seed-
lings (Gillaspy et al., 1995).

0378-1119/$ - see front matter D 2003 Elsevier B.V. All rights reserved.
doi:10.1016/j.gene.2003.09.048
36 J.C. Styer et al. / Gene 326 (2004) 35–41
These data give us information on the average expression
level of IMPs within tissues, but do not give us any
information on the spatial regulation of the genes within
tissues. Therefore, to test whether specific IMP genes are
spatially regulated in their expression, we isolated and
analyzed genomic DNA corresponding to two of the three
tomato IMP genes (LeIMP-1 and LeIMP-2). The data pre-
sented here show that the LeIMP-1 and LeIMP-2 genes differ
in their exon – intron organization. The analysis of the regu-
lation of the LeIMP-2 promoter indicates that the LeIMP-2
promoter is active in a spatially restricted subset of cells in
tomato seedlings. This suggests that LeIMP-2 plays a unique
role in inositol metabolism and recycling in tomato.
2. Materials and methods
2.1. Plant materials and treatments
Tomato Lycopersicon esculentum (cv. Ailsa Craig) seeds
were surface sterilized for 10 minutes using 30% Clorox,
rinsed with sterile water three times and germinated on
0.5  Murashige and Skoog Basal Salt Mixture media (MS
salts) (Sigma) in magenta boxes or soil (Sunshine Mix). For
Li+ and inositol treatment of plants, LiCl or inositol (Sigma)
was added to the media at 10 and 50 mM final concen-
trations, respectively. Transgenic seed were selected on the
ability to germinate in the presence of media containing 50
Ag/ml of kanamycin sulfate (Sigma). Plants were grown
either in magenta boxes at 22 jC under continuous light in a
growth chamber (Percival Scientific) or to maturity in soil in
a greenhouse.
2.2. Gene cloning and sequence analyses
Fragments of the LeIMP-1 and LeIMP-2 genes were used
to screen a Egt11 genomic library constructed from tomato
DNA (Narita and Gruissem, 1989). DNA from hybridizing
plaques was purified, cloned and sequenced as previously
described (Narita and Gruissem, 1989). The genomic
sequences of LeIMP-1 and LeIMP-2 have been deposited
into Genbank (accession numbers AY227666 and
AY227667, respectively). Genomic sequences were com-
pared to the previously published cDNA sequences of
LeIMP-1 and LeIMP-2 (Gillaspy et al., 1995) to predict
exon – intron organization boundaries using ClustalW soft-
ware (Thompson et al., 1994). Putative regulatory elements
were analyzed using the Plant Cis-Acting Regulatory Ele-
ment (Plant CARE) website (Lescot et al., 2002). Genomic
DNA from tomato plants was prepared and analyzed by
southern blotting as described (Berdy et al., 2001). IMP
amino acid sequences were aligned with ClustalW software
(gap weight = 4, gap extension weight = 0.05). Unrooted
phylogenetic trees were created with PAUP 4.0 b10 (Swof-
ford, 2002) using a parsimony algorithm based on the full
alignment. A parsimony analysis was performed with a
heuristic search of tree-bisection-reconnection branch swap-
ping. The gaps were treated as missing data. The initial
MaxTrees were set as 100 starting trees obtained via
stepwise addition. The number of trees held at each step
during stepwise addition was 5. Confidence of the group-
ings was estimated using 500 bootstrap replications.
2.3. Transgenic plant construction
Transgenic tomato plants were constructed in the follow-
ing manner: a 1.2-kb fragment containing the putative
promoter region of LeIMP-2 (LeIMP-2p) was amplified by
high-fidelity PCR utilizing oligonucleotide primers contain-
ing NcoI restriction enzyme sites. The resulting PCR frag-
ment was digested with NcoI and cloned into the NcoI site
of plasmid pSLJ4K1 which contains the open reading frame
(ORF) of the uidA (GUS) gene and nopaline synthase 3V
processing sequences (nos 3V) (Jones et al., 1992). The
position of the LeIMP-2 ( + 1) site relative to its initiating
methionine codon (ATG) was kept intact. The LeIMP-2p-
GUS-nos 3V region was removed by digestion with SstI and
BamH1 enzymes and subcloned into the binary vector
pSLJ7292 (Jones et al., 1992). This construct was trans-
ferred to Agrobacterium tumefaciens strain LBA4404 by
triparental mating, and leaf disc transformation was per-
formed using methods previously described (An et al.,
1986). Putative transformants were isolated on kanamycin
selective media and analyzed for the presence of LeIMP-2p-
uidA insertions by PCR. Transgenic plants were transferred
to soil, maintained in greenhouses and allowed to self-
fertilize. Subsequent generations of plants were obtained
from transgenic seed and maintained under standard green-
house conditions. Construction and analysis of transgenic
hydroxymethylglutaryl reductase (HMGR) plants has been
described (Jelesko et al., 1999).
2.4. h-Glucuronidase (GUS) assays
Seedling tissues were dissected using a sterilized double-
edged razor blade, immersed in GUS assay solution (0.025
AM ferrocyanide, 0.025 AM ferrous cyanide, 0.1 mM
sodium phosphate buffer pH 7.0, 0.01% Triton X-100, 0.5
Ag/ml 5-bromo-4-chloro-3-indoyl-D-glucuronic acid), vacu-
um infiltrated for 30 min and incubated at 37 jC overnight.
After incubation, the assay solution was replaced by forma-
lin – acid – alcohol (FAA: 50% (v/v) ethanol, 10% (v/v) 37%
formaldehyde, 5% (v/v) glacial acetic acid) to preserve
tissue morphology. Control transformed seedlings contain-
ing an empty pSLJ7292 vector and wildtype tomato seed-
lings were similarly treated and served as negative controls
in GUS assays.
2.5. Imaging and histology
Tissue was examined using a stereomicroscope (Stemi
SVII Apo, Ziess). Results were recorded using a digital
 J.C. Styer et al. / Gene 326 (2004) 35–41 37

(Gillaspy et al., 1993) and sectioned with a microtome

(Microm HM 330) into 10 Am sections. Tissue sections
were viewed with light or darkfield microscopy (Ruzin,
1996), utilizing a Ziess Axioscope 2 compound microscope.
Images were recorded using a Spot Digital Camera (SPOT
model 1.4.0, Diagnostic Instruments) and analyzed using
Adobe PhotoShop 5.0.

3. Results and discussion

3.1. Isolation and analysis of the genomic structure of the

LeIMP-1 gene

To characterize the genomic structure of the LeIMP-1

Fig. 1. Genomic blot of tomato DNA with LeIMP-1 and LeIMP-2 probes.
Tomato seedling genomic DNA was digested using HindIII, EcoRI or
BamHI, separated on an agarose gel and analyzed by Southern blotting with
LeIMP-1 and LeIMP-2 probes as described in Section 2.
camera (3CCD Camera, MIT) and prepared for publication
using Adobe PhotoShop 5.0. Tissue sections were obtained
by dehydration and infiltration with paraffin as described
gene, we used an LeIMP-1 cDNA probe to screen a
tomato genomic DNA library as described in Section 2.2.
This probe hybridizes to a unique pattern of bands in a
tomato genomic southern blot (Fig. 1). A genomic clone
containing 5868 bp of DNA corresponding to the LeIMP-
1 gene resulted. By aligning the LeIMP-1 genomic
sequence (Genbank accession number AY227666) to the
nucleic acid sequence of the LeIMP-1 cDNA (Genbank
accession number U39444), we found that the LeIMP-1
ORF consisting of 273 amino acids is encoded by 12
exons spanning 3646 bp of DNA (Fig. 2A). We also
obtained 1401 bp of the LeIMP-1 promoter sequence and
116 bp of the 5VUTR sequence. Analysis of the promoter
region using the internet sequence analysis tool, Plant
CARE (Lescot et al., 2002), identified a TATA box at
( 25) and many putative light regulatory elements
(Manzara and Gruissem, 1988; Terzaghi and Cashmore,
1995). Published data indicates that the putative light
regulatory elements function in the regulation of tran-
scription of LeIMP-1, as both LeIMP-1 mRNA and

Fig. 2. Genomic organization of the LeIMP-1 and LeIMP-2 genes. (A) The LeIMP-1 gene. Black boxes represent exons; sizes (in bp) are in black below the
boxes. Intron sizes (bp) are indicated in gray. Start and stop refer to the positions of the start and stop codons, respectively. (B) The LeIMP-2 promoter and gene.
The large black box represents the single exon. Positions of the TATA box, transcriptional start site ( + 1), start and stop codons are indicated by arrows. Patterned
boxes (I – IV) represent putative transcriptional regulatory regions. Regions I, III and IV: light-responsive elements, region II: cold responsive element.
38 J.C. Styer et al. / Gene 326 (2004) 35–41
protein levels increase in seedlings exposed to light
(Gillaspy et al., 1995).
3.2. Isolation and analysis of the genomic structure of the
LeIMP-2 gene
To characterize the genomic structure of the LeIMP-2 gene,
we screened our tomato genomic DNA library with the
LeIMP-2 cDNA probe which we had also used in a Southern
blot of tomato genomic DNA (Fig. 1). Sequencing of a
positive clone revealed congruity between the observed pat-
tern of hybridization in the Southern blot and the genomic
clone. We aligned the LeIMP-2 genomic DNA sequence
(2726 bp, Genbank accession number AAY227667) with
the LeIMP-2 cDNA (Genbank accession number U39443).
This alignment revealed that the LeIMP-2 gene contains a
single, uninterrupted ORF of 798 bp (Fig. 2B). The absence of
introns is unique to LeIMP-2, as PCR amplification using
LeIMP-3-specific oligonucleotide primers with tomato ge-
nomic DNA confirmed the presence of introns in parts of the
LeIMP-3 gene (data not shown). The absence of introns is rare
in plant genes; however, intron-less genes have been docu-
mented (Hossain et al., 1996; Lui et al., 1997). Many chloro-
plastic and mitochondrial genes have few or no introns,
possibly due to their prokaryotic origins. It is possible that
transposon insertions into the tomato genome may have
provided the organism with an intron-less copy of one of the
LeIMP genes. Within the 2726 bp of DNA obtained at the
LeIMP-2 locus, we also identified 338 bp of DNA that is
identical to the 3V end of a tomato calmodulin-like gene
(Genbank accession number BG130697). Calmodulin, a
148-amino acid protein, binds calcium and functions to
activate regulatory subunits of other enzymes such as calmod-
ulin-dependent protein kinases (Luan et al., 2002). Down-
stream of the calmodulin-like gene, we identified 1,205 bp of
DNA of the putative LeIMP-2 promoter. Analysis of this DNA
revealed the presence of a putative TATA box at ( 30) and a
predicted start site of transcription 114 bp upstream from the
start site of translation and four putative regulatory elements
were found in this promoter sequence (I through IVin Fig. 2B).
We previously reported that light stimulated an increase in
LeIMP-2 mRNA expression, so it is likely that these elements
I, III and IV are functional (Gillaspy et al., 1995).
3.3. Comparison of the genomic structure of plant and
animal IMP genes
To determine if other plants contain an IMP multigene
family, we analyzed the genome of Arabidopsis thaliana for
IMP homologues. By using ClustalW and PAUP* software,
we determined that the Arabidopsis genome contains a
single IMP gene (AtIMP; At3g02870) with a conserved
exon – intron organization to LeIMP-1, and two distantly
related IMP-like genes (At1g31190 and At4g39120), which
are predicted to contain nine exons with no apparent exon–
intron organizational conservation to the AtIMP or LeIMP
genes. ClustalW alignments and PAUP* phylogenetic anal-
yses revealed that At1g31190 and At4g39120 are more
similar to the SuhB gene (Chen and Roberts, 2000; Matsu-
hisa et al., 1995) found in both prokaryotes and eukaryotes
than to IMP genes. In comparing the plant IMP genes with
the mouse and human IMP genes, we found that both types
of animal genes exhibit identical exon– intron organization
and this structure differs from that of the plant IMP genes.
To address whether multiple IMP genes are likely to have
evolved through a gene duplication event, we constructed a
phylogenetic tree of tomato, Arabidopsis, mouse, human,
dictyostelium and yeast IMP proteins (Fig. 3). The branch-
ing pattern of human and mouse IMP sequences indicates
that the two animal IMP genes arose through a gene
duplication of an ancestral gene. In contrast, both the yeast
and plant IMP gene families are most likely a result of
separate gene duplication events. In particular, plant IMP
gene duplication events occurred after the divergence of
Arabidopsis and tomato, one of which produced an intron-
less copy (LeIMP-2).
3.4. Spatial regulation of the LeIMP-2 promoter
We previously reported that LeIMP-2 mRNA expression
was very low in all tomato tissues examined by RNAse
protection assays (Gillaspy et al., 1995). Since RNAse
protection analysis gives information on the average expres-
sion level in tissues and does not provide spatial information,
we determined whether the LeIMP-2 gene was expressed in a
subset of cells. The 1.2-kb LeIMP-2 promoter (LeIMP-2p)
Fig. 3. Phylogenetic analysis of IMP proteins. Amino acid sequences were
aligned using ClustalW (Thompson et al., 1994; gap weight = 4, gap
extension weight = 0.05). Alignments were analyzed with PAUP 4.0 b10
(Swofford, 2002) to generate an unrooted tree. Confidence of the groupings
was estimated using 500 bootstrap replications. Bootstrap values present at
each node represent the percent of times out of 500 bootstrap resamplings
that branches were grouped together. Branch lengths are to scale. Genbank
accession numbers for IMPs from tomato (LeIMP-1, LeIMP-2, LeIMP-3):
P54926, P54927, P54928; from mice MmImpa1, MmImp2a): AAB97469,
AAK39516; from humans (Hs IMPA1, HsIMPA2): P29218, O14732; and
yeast (ScInm1, ScInm2): AAB68918, AAB64472; and from Arabidopsis
(At3g02870): AAF26973. Dictyostelium discoideum (DdIMP) is from the
Dicty Database (genef00868).
 J.C. Styer et al. / Gene 326 (2004) 35–41 39

was fused to the uidA gene encoding GUS and transferred
into tomato plants by A. tumefaciens-mediated transforma-
tion (An et al., 1986). Analysis of homozygous T2 transgenic
seedlings from four independent lines indicated that the
LeIMP-2 promoter directed expression of GUS predomi-
nantly in the stem/leaf junction of the seedling stem (Fig. 4).
The most intense deposition of GUS product was found
below the shoot apex and extended on the abaxial side of the
petiole of the first 1 – 2 sets of expanded leaflets into two
abaxial ‘‘stripes’’ (Fig. 4B,C). In addition, other tissues (i.e.
stems, leaves, flowers) from mature plants and plants trans-
formed with an empty vector did not contain appreciable
amounts of GUS activity. To determine specific cell types in
which the LeIMP-2 promoter was active, we examined
preserved sections of 21 day old transgenic LeIMP-
2p:GUS seedlings stained for GUS activity (Fig. 4G,H).
From these sections, we conclude that LeIMP-2 expression is
strongest in the epidermal and first few cortex cell layers of
the stem and petiole (Fig. 4G – H). Surprisingly, cells within
the shoot apical meristem and developing leaf primordia also
showed LeIMP-2p:GUS expression (Fig. 4G).
The significance of the LeIMP-2p:GUS expression pat-
tern is not immediately obvious. One can speculate that
genes exclusively expressed at stem/leaf junctions might be
regulated by a type of ‘‘nutrient eddy’’ present at this
location. For example, the hydroxymethylglutaryl Co-A
enzyme reductase 1 (HMGR-1) gene from tomato is
expressed in a similar location, although the abaxial
‘‘stripes’’ of LeIMP-2 expression are unique (compare Figs.
4B and 5D). Since a role for IMP in regulating dorsal –
ventral specification in Xenopus laevis has been studied
(Busa and Gimlich, 1989; Maslanski et al., 1992), it is
intriguing to speculate that the LeIMP-2 gene in tomato
might play a similar role in stem/leaf abaxial specification.
3.5. Regulation of the LeIMP-2 promoter by inositol and
Li+
We were interested in whether the product of IMP
catalysis, inositol or an inhibitor of IMP catalysis, Li+,
regulate the LeIMP-2 promoter. To address this, we grew
LeIMP-2p:GUS seedlings for 21 days in the presence of

Fig. 4. Spatial regulation of the LeIMP-2 Promoter. The LeIMP-2 promoter was fused to the uidA reporter gene and transformed into tomato. Twenty-one-day-
old transgenic seedlings were assayed for GUS activity as described in Section 2. (A) Control, non-transformed seedling shoot apex, compound leaf and stem.
Bar = 1 mm. (B – H) LeIMP-2p:GUS seedlings. (B) Shoot apex, compound leaf and stem. Bar = 0.5 mm. (C) Close-up of stem and leaf. Bar = 2 mm. (D)
Adaxial internode surface. Bar = 2 mm. (E) Side surface of internode. Bar = 2 mm. (F) Abaxial surface of internode. Bar = 2 mm. (G) Dark-field microscopy of
a longitudinal section of the shoot apical meristem and leaf primordia. Bar = 0.14 mm. (H) Dark-field microscopy of a close-up of a transverse section of the
base of the shoot apex. Bar = 0.10 mm.
40 J.C. Styer et al. / Gene 326 (2004) 35–41

Fig. 5. Regulation of the LeIMP-2 promoter by inositol and Li+. (A – C) Twenty-one-day-old LeIMP-2p:GUS seedlings were grown on media alone (A), media
plus 50 mM inositol (B), or 10 mM LiCl (C), and assayed for GUS activity as described in Section 2. (D – F) Twenty-one-day-old HMGR-1p:GUS seedlings
were grown on media alone (D), media plus 50 mM inositol (E), or 10 mM LiCl (F), and assayed for GUS activity as described. Bar = 1.4 mm.

control media, media supplemented with 50 mM inositol, or
media containing 10 mM LiCl. We have previously char-
acterized the effect of LiCl on tomato seedling development.
Our results indicated that LiCl takes 42 days to disrupt
development of the stems and leaves (Gillaspy and Gruis-
sem, 2001). Therefore, growth for 21 days in the presence of
either LiCl or inositol does not outwardly affect develop-
ment. These treatments do, however, affect LeIMP-2 ex-
pression (Fig. 5). Inositol supplementation decreased overall
GUS staining in both the stem/leaf junction and in the
abaxial ‘‘stripes’’ that were present in untreated seedlings
(Fig. 5A,B). Addition of LiCl reduced almost all visible
GUS staining (Fig. 5C). To determine if the effects of
inositol and LiCl were specific to the LeIMP-2 promoter,
we also examined HMGR-1p:GUS plants grown under
similar conditions. Neither inositol nor LiCl treatment
decreased expression of the HMGR-1 promoter (Fig. 5D –
F). Therefore, we conclude that inositol and LiCl act
specifically to reduce LeIMP-2 expression.
The yeast Saccharomyces cerevisiae contains two IMP
genes (INM1 and INM2); the INM1 gene has been shown to
be upregulated by inositol and downregulated by Li+ (Mur-
ray and Greenberg, 1997, 2000). The authors of this study
speculate that INM1 expression is tied to the need for
recycling inositol, which is a breakdown product of PIP
signaling molecules. Thus, when cellular synthesis of PIP is
high, the concentration of its breakdown product, inositol,
will be increased. Conversely, since Li+ inhibits IMP and
decreases the amount of inositol in yeast (Murray and
Greenberg, 2000), Li+ treatment could decrease INM1 ex-
pression because of a decreased need for inositol recycling.
We found a similar decrease in LeIMP-2 expression in the
presence of Li+. However, our results showing inositol also
decreases LeIMP-2 expression indicates a difference in IMP
gene regulation between yeast and plants. We speculate that
inositol inhibition of LeIMP-2 expression in plants may
indicate that this specific IMP isoform is regulated by
metabolic feedback from its product, inositol. We further
 J.C. Styer et al. / Gene 326 (2004) 35–41
speculate that plants, which channel inositol and its break-
down product, glucuronic acid, into a variety of metabolites,
contain a more complex regulation of IMP expression. First, a
multiple gene family may allow for both spatial and devel-
opmental controls, which gives the plant differential ability to
synthesize and recycle inositol in different tissues. Second,
our results here indicate that both inositol and Li+ can alter
expression of at least one of these gene family members.
3.6. Conclusion
 The genomic sequences of two of the three tomato genes
(LeIMP-1 and LeIMP-2) encoding myo-inositol mono-
phosphatase have been determined.
 The LeIMP-1 gene contains 12 exons and the exon –intron
organization is conserved in the single A. thaliana (At)IMP
gene. In contrast, the LeIMP-2 gene contains no introns
and may have arisen from an integration of a retroviral
copy of the LeIMP-3 gene in the tomato genome.
 The LeIMP-2 promoter directs the expression of the
LeIMP-2 gene in a spatially restricted pattern. In tomato
seedlings, expression of the LeIMP-2 gene is observed in
the stem/leaf junction and in abaxial ‘‘stripes’’ on the
stem and in the petiole of developing leaves.
 LeIMP-2 promoter activity is downregulated by both the
product of IMP catalysis, inositol and an inhibitor of
IMP, Li+. Differences between yeast and plants with
regard to regulation by inositol may reflect a more
complex regulation of inositol utilization by plants.
Acknowledgements
The authors would like to thank Susan Jenkins for help in
construction of transgenic plants, JinRi Shi and John Jelesko
for preliminary characterization of transgenic plants, Jake
Tu and Hongguang Shao for help in the phylogenetic
analysis, and Bhadra Gunesekera for comments on the
manuscript. This work was supported by a Jeffress
Memorial Trust award and the Commonwealth Health
Research Board of Virginia.
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