We are unable to create an online viewer for this document. Please download the document instead.
Electronic Journal of Biotechnology ISSN: 0717-3458 Vol.7 No.1, Issue of April 15, 2004
© 2004 by Pontificia Universidad Católica de Valparaíso -- Chile Received September 28, 2003 / Accepted February 25, 2004
Isolation and study of a ubiquitously expressed tomato pectin
methylesterase regulatory region
Departamento de Tecnología de Alimentos de Origen Vegetal
Centro de Investigación en Alimentación y Desarrollo, A.C.
Carretera a la Victoria km. 0.6
Apartado Postal 1735
Hermosillo, Sonora, 83000, México
Tel: 52 662 80 00 55
Fax: 52 662 280 04 22
Genetique Moleculaire des Plantes
Universite Joseph Fourier
Cermo BP 53. 38041 Grenoble Cedex 9, France
Fax: 33 4 76 51 43 36
Tel: 33 4 76 51 44 41
Avtar K. Handa
Department of Horticulture and Landscape Architecture
1165 Horticulture Building
West Lafayette, IN, 47907-1165, USA
Tel: 765 494-1339
Fax: 765 494-0391
Keywords: pectin Methylesterase, promoter analysis, tobacco transgenic plants, tomato.
Pectin methylesterase (PME) is an enzyme located in the
Pectin methylesterase (PME) is an enzyme that have been
plant cell wall of higher plants whose physiological role
found in every plant tissue analyzed (Lineweaver and
is largely unknown. We had isolated a PME gene from a
Jansen, 1951; Rexova-Benkova and Markovic, 1976),
tomato genomic library, including 2.59 kb of 5´ flanking
several fungi (Christgau, et al. 1996; Mendgen, et al. 1996),
region and the coding region. Both coding and promoter
bacteria (Plastow, 1988; Barras et al. 1994) and even
region were sequenced and computer analyzed. Tobacco
insects (Ma et al. 1990; Shen et al. 1999). In higher plants,
transgenic plants were created harboring constructs in
it is known to be a cell wall associated protein and several
which 2.596 Kb, 1.306 Kb and 0.267 Kb sizes of the
of the PME cDNA available in the literature, are known to
promoter were driving the expression of ?-
have toward the N-terminal sequence, a characteristic
Glucuronidase gene (GUS). GUS activity was studied by
signal peptide which is thought to help in targeting the
histochemical and fluorometric assays. Two introns of
protein to the plant cell wall (Gaffe et al. 1997 and
106 and 1039 bp were found in the coding region and
references therein). PME catalyzes the deesterification of
phylogenetic analysis placed this PME gene closer to
galactosyluronate methylesters of pectins, releasing protons
genes from Citrus sinensis and Arabidopsis thaliana than
and methanol into the media (Frenkel et al. 1998). Despite
tomato fruit-specific PME genes. In the promoter, it was
the biochemical mode of action of PME is well known, it
found direct repeats, perfect inverted repeats and light
have been difficult to demonstrate any role for PME in the
responsive elements. GUS histochemical analysis physiology of plants. However, several hypothesis had been
showed activity in all plant tissues with the exception of
proposed: pollen germination and/or tube growth (Mu et al.
pollen. The reduction in the promoter size induced a
1994), abscission (Sexton and Roberts, 1982), regulation of
reduction in GUS activity in root, stem and leaf. cell enlargement through changes in the plant cell wall
Furthermore, root and leaf showed the highest and
Donnan potential (Ricard and Noat, 1986), fruit softening
lowest activity, respectively. We had isolated a tomato
during postharvest fruit ripening (Zeng et al. 1996) and
PME gene with novel characteristics as compared with
plant defense (Chamberland et al. 1991; Wietholter et al.
other known PME genes from tomato.
2003). Furthermore, strong experimental evidences had
This paper is available on line at http://www.ejbiotechnology.info/content/vol7/issue1/full/1
Tiznado, M. E. et al.
been provided to suggest that a PME role in the release of
plaques producing signal in both lifts were chose to
cells from the root cap (Stephenson and Hawes, 1994; Wen
continue. After four rounds of purification and screening,
et al. 1999), plant pathogenesis (Collmer and Keen, 1986;
one plaque turns out to be positive. Elimination of the
Mendgen, et al. 1996; Nun et al. 1996; Valette-Collet et al.
bacteria present in the agar was made by using chloroform-
2003), plant systemic infection by tobacco mosaic virus
containing SM buffer. For phage amplification, E. coli
(Chen and Citovsky, 2003) and maintenance of the tomato
strain LE 392 was infected with the phage after cultured in
fruit tissue integrity during postharvest shelf life (Tieman
LB media. DNA isolation from the phage was made
and Handa, 1994). However, the actual physiological role
through a phenol chloroform protocol (Ausubel et al. 1988).
of PME is still matter of controversy.
Digested DNA with several restriction enzyme was
separated by electrophoresis and blotted into nylon
Four papers to our knowledge had been published in which
membranes. These DNA blots were probed with the PME
the cloning of regulatory sequences of PME from higher
cDNA complete sequence available to locate the phage
plants was described. However, in all of them DNA DNA region encoding the genomic PME gene and the
comparison by computer was the only tool used to prove
region 5´ upstream. Sal I and EcoR I digested DNA
that the gene located downstream was indeed encoding a
fragments were subcloned into pBSKS (+/-) vector
pectin methylesterase. Albani et al (1991) reported the
(STRATAGENE CLONING SYSTEMS. La Jolla, CA).
finding of a genomic clone from Brassica napus which
contains the PME gene and its 5´ upstream regulatory
The EMBL3A ? phage library screened was created using
region. Studies were conducted using a piece of the gene
the Sal I restriction site. Because of this, digested DNA
located dowstream as a probe. This gene was found to be
fragments using Sal I were used to calculate the size of the
expressed mainly during pollen development. Two putative
tomato DNA inserted into the phage isolated, found to be
PME promoter regions were cloned from Brassica
13.7 kb. All the procedure above mentioned was performed
campestris (Kim et al. 1997). Study of their sequence
essentially as described (Sambrook et al. 1989) unless
found them to have high homology with the previously
reported promoter PME from Brasicca napus (Albani et al.
1991). Further, a sequence motif similar to the one known
DNA sequencing of the promoter and the genomic
to exist in two tomato pollen-specific promoter was located.
Tobacco transgenic plants with constructs containing two
different promoter sizes from one of those two promoter
Nested unidirectional deletions of the 5´ upstream DNA Sal
available were made. Expression of the GUS gene was
I fragment were made by following the recommendations
only detected during developing and mature pollen grains
of the company (Erase-a-Base® System, Promega
germinated in vitro. Recently, the cloning of two 5´ Corporation, Madison, WI). Deleted clones with about 250
upstream region of PME from Citrus sinensis was bp of size difference were used for DNA sequencing using
published. Northern blot analysis showed that both DNA
the T3 universal primer by the Sanger dideoxy chain
regulatory region are active in most of vegetative tissues
termination technique following the recommendations
(Nairn et al. 1998).
(Sequenase Kit, United States Biochemical Corporation,
Cleveland, Ohio). Second strand sequencing was
In our laboratory, we have cloned a 13.7 kb. genomic DNA
determined by the DNA sequencing facility of IOWA State
from tomato containing a 2.59 kb. of DNA 5´ flanking
University by using primers designed at proper positions in
region, along with all the PME genomic clone. the sequence (Iowa State University, Ames, Iowa).
Identification of the protein encoded by the gene
downstream was made by creating tobacco transgenic Creation of the constructs
plants over expressing the PME cDNA (Gaffe et al. 1997)
and comparing the sequences of the genomic and cDNA
Three chimeric constructs driving the ?-glucuronidase gene
regions. In this work, we describe the study of a 5´ flanking
(uidA) under different sizes (2.596 Kb, 1.306 Kb and 0.267
region of a PME gene called pmeu1 (which stands for PME
Kb) of the promoter region were created by transcription
ubiquitous one) using computer tools and tobacco fusion through the insertion of two stop codons in between
the ATG of the pmeu1 gene and the ATG of the uidA gene.
Every chimeric construct was ligated into the promoterless
MATERIALS AND METHODS
binary vector pBI101.3 (Bevan, 1984). This plasmid
includes the neomycin phosphotransferase gene (NPTII)
Cloning of the genomic fragment
which confers resistance to kanamycin to be used as
selectable marker. Furthermore, in this plasmid the DNA
The plaque lift technique was used to screen 810,000 clones
introduced is located between the right and left borders of
of a tomato cv ‘Cherry’ genomic library made in EMBL3A
the T-DNA, which allows the transference into the plant
? phage with a radiolabeled small piece of PME; cloned by
genome by Agrobacterium infection (Hooykaas, 1989;
RT-PCR from tomato roots poly-A+ mRNA (Gaffe et al.
Zupan and Zambryski, 1995; Nester et al. 1996). Proper
1995). We found several hybridizing, putatively positives,
insertion of the different promoter sequences into the
plaques. From every plate, we made two lifts and only that
plasmid was confirmed by DNA digestion using suitable
Isolation and study of a ubiquitously expressed tomato pectin methylesterase regulatory region 11
restriction enzymes and PCR using primers designed changed by the GUS staining solution and left at 37ºC for at
against sequences in the PME promoter and uidA gene.
least 18 hrs before examination for GUS staining.
Every chimeric construct created included 150 bp in Computer analysis
between the ATG of the pmeu1 gene and the ATG of the
uidA gene, containing sequences from the pmeu1 gene and
DNA sequence from the different pmeu1 5´ flanking region
pBSKS(+/-) phagemid and pBI101.3 binary vector. deletions were joined together using DNAsis (Hitachi
Sequencing between the two ATG’s was used to verify the
Software Engineering Co., LTD., 1991). Comparison
presence of two stop codons and to corroborate the between the pmeu1 cDNA and pmeu1 genomic clone was
transcription fusion of the two ATG.
performed using Harr plot analysis with DNAsis software.
Presence of known cis-acting elements was determined
Tobacco transgenic plants
using the programs MathInspector ver. 2.2 (Quandt et al.
1995), TFSEARCH ver. 1.3 (Parallel Application, Tsukuba
Mobilization of the pBI101.3 plasmid into Agrobacterium
Laboratory, RWCP, Japan), Signal Scan ver. 4.05
LBA4404 was performed by triparental mating using the
broad-host helper plasmid pRK2013 (Ditta, 1981). (Prestridge, 1991) and Pattern Search (Wingender et al.
Agrobacterium transconjugants were screened on plates
1996; Wingender et al. 1997). Percent of identity among the
containing a mixture of kanamycin and rifampicin different PME promoters and PME transcribed regions
antibiotics in YEP media (Sambrook et al. 1989). were determined using Align (Myers and Miller, 1988).
Verification of the mobilization of the constructs was made
Alignment of deduced amino acid sequences was
by purification DNA from Agrobacterium following the
performed using GCG’s Pileup Program (Genetics
recomendations (Wizard Minipreps, Promega Corporation,
Computer Group, Madison, WI). Multiple sequence
Madison, WI) and digestion with proper restriction alignment was performed using CLUSTAL W (Thompson
enzymes. Tobacco (Nicotiana tabaccum W38) young et al. 1994). DNA direct repeats for the tomato PMEU1
leaves were infected with Agrobacterium by using the leaf
promoter were determined using Proscan ver 1.7 and
disk technique (Mathis and Hichee, 1994) and selection for
repeats from GCG software ver. 9.0 (Genetics Computer
transformants was done by using kanamycin in the media.
Group, 1995). Perfect inverted repeats (mirror repeats) were
located using Palindrome from GCG software ver 9.0
GUS activity measurement
(Genetics Computer Group, 1995). Putative TATA box was
located by Signal Scan ver. 4.05. Phylogenetic analysis
After induction of roots, about 50 primary independent
were done using the phylogeny inference package
transformant plants growing in vitro harboring every of the
(Felsenstein, 1989; Felsenstein, 1993).
three constructs were selected at random to measure GUS
activity in leaf. This was done using the fluorometric Statistical analysis
technique (Jefferson et al. 1987) with a Perkin Elmer LS5
fluorometer. Quantification of reaction product was done
Comparison of leaf GUS activities for the three constructs
by using a 4-methylumbelliferone standard curve. Also, six
and for the different tissues was made by variance analysis
independent transgenic plants were used to measure GUS in
using a completely randomized design for unbalanced
root, stem and leaf tissues. Every GUS measurement was
number of repetitions. Tukey test was used when needed to
done at least three times. For enzymatic specific activity,
find differences among means. Because it is known that the
protein determination was made using Bradford (1976) with
GUS enzymatic activity in populations of first-generation
bovine serum albumin as standard.
transgenic plants does not follow a normal distribution
In order to examinate the GUS presence in different tissues,
(Nap et al. 1993), we performed a Box-Cox transformation
at least 20 primary transgenic plants harboring the different
before variance analysis. From here, we learned that a
constructs, were vacuum infiltrated with a 1.9 µM solution
square root was a suitable transformation to bring the GUS
of 5-Bromo-4-Chloro-3-Indolyl-Glucuronide (Jefferson et
activity parameter into normality. Statistics reported in this
al. 1987) as described (Mandel et al. 1995).
paper represents the back transformation of the square root
transformed data. All statistical analysis were performed
Pollen germination in vitro
using the SAS software (SAS Institute Inc. Cary, N.C.).
Tobacco flowers in anthesis were collected from plants
growing in the greenhouse and transported immediately to
the laboratory. Anthers were cut and only that pollen Isolation and characterization of PMEU1 gene
released by a gently shaking was used for germination
studies. Pollen was germinated using the Brewbaker and
The cloning and characterization of the entire PMEU1
Kwack solution as described (Brewbaker and Kwack, tomato cDNA has been previously reported (Gaffe et al.
1963). Histochemical GUS staining was performed after
1996; Gaffe et al. 1997). The next step lead us to the
four hours of pollen germination. Germination solution was
isolation and characterization of the genomic fragment
Tiznado, M. E. et al.
containing the PMEU1 gene. An EMBL3A phage of a
The position of one intron, relative to the deduced amino
tomato genomic library (VNTF cherry) was screened using
acid sequence, is conserved in 19 out of the 22 plant PME
300 bp cDNA fragment corresponding to the conserved
genomic sequences. This intron is located 17 amino acid
PME domain in PMEU1 (Gaffe et al. 1996; Gaffe et al.
residues upstream of the PME signature sequence
1997). Four rounds of phage amplification allowed us to
GPXKHQAVALR; observed in the rice genomic clone as
purify a single positive clone.
well (Figure 3). This observation suggest that monocots and
dicots share a common ancestor. The other three clones
Subcloning, analysis by restriction mapping and DNA blot
(AtPME8, AtPME9 and AtPME10) are clustered together
of the tomato genomic DNA fragment contained in the
in one group by the phylogenic analysis (Figure 4) which
EMBL3A phage indicated that the size of the inserted
agrees with the lack of the intron located at the same
tomato genomic DNA is 13.7 kb and the PMEU1 gene was
distance from the signature sequence and the common
found to be located toward the 5´ region, spanning 5.28 kb.
characteristic of the presence of four introns.
In Figure 1, is presented the organization of the EMBL3A
Phylogeny analysis among PMEU1 and other plant
clone containing the PMEU1 gene This region includes
2.59 kb of DNA regulatory region and 2.89 kb of DNA
transcribed region, shown as white and black areas. In the
Deduced amino acid sequences of 22 plant PME genes as
figure it is also shown the location of the right and left
well as PMEB from Erwinia chrysanthemi were included in
lambda phage arms and the main restriction sites.
our study. The plant PME genes were chosen based in
published data providing experimental evidences or
DNA sequence of the transcribed region of PMEU1
presence of the full genomic sequence from the Arabidopsis
thaliana genome project from which some of the PME
genes were included. One of the pectin methylesterase
In Figure 2, it is shown the sequence of the PMEU1
genes from Oriza sativa was included to be able to compare
genomic clone (GenBank Accession Number: AY046596).
with a PME from monocots. Furthermore, the gene from E.
In italics, it is presented the 5´ untranslated region (Gaffe et
chrysanthemi was chosen in order to compare PME from
al. 1997) and the partial 3´ untranslated region. In bold, it is
plants with a distantly related PME and also to have a
shown the sequence of the two introns present. Underlined,
control in the phylogenetic analysis. The PMEU1 gene
it is presented the translation start site and stop codon
includes 2900 bp and a theoretically deduced open reading
(TAA). Double underlined it is shown the putative frame of 583 amino acids (Figure 3). Several sequences
polyadenylation signal and polyadenylation site (GT).
shorter than 400 bp like PECS-1-2 from Citrus sinensis, are
known to be partial. However, PPE1 sequence from
The polyadenylation signal was found to follow the plant
Petunia inflata is shorter than 400 bp and still encodes a
consensus sequence AAUAAA (Li and Hunt, 1995). The
two introns present are of 106 and 1039 bp in length. Both
of them showed a significantly higher composition of U’s
Sequence alignment of these different encoded
with respect to the flanking exon sequences. This is a
polypeptides indicate that the N-terminal half of these
characteristic known to be present in many plant genes (Ko
clones is loosely conserved compared with the C-terminal
et al. 1998).
half, involved perhaps in the PME catalytic activity (Figure
3). Because of this, a final alignment, edited to represent
Intron-exon lrganization of PMEU1 and other PME
only the phylogenetically relevant fraction of the sequences
was used to derived a phylogenetic tree (Figure 4).
The intron-exon structure of the PME genomic sequences
Based on this phylogenetic analysis, we organized up to 18
available has been analyzed. The splice junction of all the
genomic clones in five groups. Five PME genomic clones
clones conform to the GT/AG boundary rule for the 5´
from various origins can not be associated with any of these
donor and 3´ acceptor site (Liu and Filipowicz, 1996). The
groups. The lack of association of PME from Erwinia
intron size range from 72 to 1577 bp and the exon from 117
chrysanthemi with other plant PME’s was something
to 1353 bp. The average value for intron and exon size is
expected, however, it is interesting that the clone PECS-2.1
109 and 519, respectively.
from Citrus sinensis is distantly related with the two clones
PECS-1.1 and PECS-1.2 from the same source that
Seventeen clones have only one or two introns. Three
clustered together with the PMEU1 clone.
putatives PME genomic sequences from Arabidopsis
contains four introns and show a level of similarity with
This phylogenetic analysis indicates that PMEU1 belong to
PMEU1 of around 50%. Further, AtPME7 with five introns
a group containing two Citrus sinensis PME genes, PECS-
is more closely related to PMEU1 (64.9% of similarity).
1.1 and PECS-1.2 and two Arabidopsis thaliana genes,
These observations suggest that there is not a simple AtPME2 and AtPME3; however, it is distant from the three
relationship between the phylogenic distance and intron
tomate PME genes expressed only in tomato fruit tissues:
number in the different PME genomic clones.
LePME1, LePME2 and LePME3 (Harriman et al. 1992),
Isolation and study of a ubiquitously expressed tomato pectin methylesterase regulatory region
suggesting the PMEU1 is a gene evolved to have a different
promoter is largely theoretical and experimental evidences
and novel function. However, due to the limited amount of
to confirm any function of these sequences remains to be
information concerning the expression of these genes, we
can not establish a clear relationship between these groups
of PME genes and their possible function.
We were able to locate a putative TATA box 44 bp
upstream of the transcription start site (Figure 5). However,
Structure of PMEU1 promoter
as mentioned for the other elements above described, the
confirmation of this region as actual TATA box still need to
In Figure 5 it is shown the 2.59 kb. PMEU1 promoter
be experimentally probed. We did not find the presence of a
sequence (GenBank Accession Number AY050764). CAAT box, although it had been shown to be present in
Computer study of this sequence showed several features
several promoter of plant genes (Joshi, 1987).
commonly present in DNA regulatory sequences. The
largest direct repeats within the promoter sequence, are
Paired comparisons among the DNA sequence of the
shown underlined and numbered. Mirror repeats are shown
PMEU1 promoter with sequences of PME promoters from
with arrows in opposite directions. Putative cis-acting Brassica campestris (GBAN215-6 and GBAN215-12),
elements are shown boxed and roman numbered. The Brassica napus (Bp 19), Citrus sinensis (CsPME1 and
putative TATA box is shown double underlined. In bold, it
CsPME3) and Arabidopsis thaliana (AtPME1) did not
is shown the transcription start site.
showed any special pattern or similarity with any of the
promoters included in the analysis. Indeed, all the pair
Study of the 5´ region of this sequence did not indicate the
comparisons showed around 50% of identity. Further,
presence of elements commonly present in the 3´ region of
analysis by multiple sequences alignment among all PME
genes, suggesting that the PMEU1 promoter region could
promoters failed to locate an homologous region in
be larger than 2.59 kb.
common to all of them (data not shown).
The number of direct repeats located by computer in the
Transgenic tobacco plants
PMEU1 promoter varied with the size of the fragment, in
such a way that it was found only one for repeats consisting
With the goal to test whether the 2.59 kb. DNA region
of 17 and 26 bp, four for repeats with 12 bp, three for
located in the 5´ flanking region of the PMEU1 genomic
repeats with 11 bp and greater than 1000 for repeats with 5
coding region represent an active promoter, we created
bp (data not shown). However, the significance of this
several tobacco transgenic plants expressing chimeric
repeats within the PMEU1 promoter remains to be constructs in which 2.59, 1.3 and 0.267 kb of promoter
sequence is driving the expression of the reporter gene uidA
encoding the ?-glucuronidase enzime.
We also locate in the promoter sequence several perfect
inverted repeats o mirror repeats, depicted in Figure 5 as
In Figure 6 it is shown the three constructs made along with
arrows pointing in opposite directions. It is interesting that
the average of leaf GUS activity for about 50 independently
the longest inverted repeats is contained within the longest
tobacco transformed plants growing in vitro and expressing
direct repeats. As in the case of the direct repeats, the
the corresponding construct. From the graph, it is clear the
function of these inverted repeats, if any, is unknown.
trend: the bigger the piece of the promoter, the higher the
activity of the uidA gene. Statistical analysis of root
Short sequences with resemblance to known cis-acting squared-transformed data found differences among all of
elements present in other ADN regulatory regions were
located in the PMEU1 promoter sequence. In Figure 6 are
included only the ones with the highest degree of similarity.
Histochemical staining of many independent primary
Two copies of the sequence GAAAGA shown to confer
tobacco transgenic seedlings showed activity in leaf, stem
responsiveness to red light in the phytochrome A3 promoter
and roots of the plants. We also found activity in petals and
(Bruce et al. 1991) are present in PMEU1 promoter (box I).
sepals. However, no activity was detected in pollen grain or
Also, one copy similar to the sequence in vitro germinated pollen (data not shown).
GTGAGGTAATAT, known to be regulated by light (Fluhr
and Dankekar, 1986; Green et al. 1987) was found (box II).
In Figure 7, it is shown the average values of GUS activity
Furthermore, we found regions similar to a G-box (box III),
for root, stem and leaf of six independent tobacco plants
shown to be light inducible (Schindler et al. 1992). Also, it
harboring every of the three constructs. The effect of
was located a sequence similar to an abscisic acid reducing the size in the PMEU1 promoter for the different
responsive element (box IV) (Guiltinan et al. 1990). As can
tissues analyzed followed the pattern already observed in
be seen from above, three of the four putative cis-acting
leaf. The decrease in the size of the PMEU1 promoter
elements located are known to be regulated by light. region reduce its transcriptional activity in all differentiated
Experiments to show whether PMEU1 promoter is tissue analyzed.
regulated by light deserves further attention. However, still
the function of this cis-acting elements within the PMEU1
Statistical analysis found significant differences (p<0.05)
Tiznado, M. E. et al.
among the root tissues from plants harboring the different
PME genes: GPXKHQAVALR. Also, we noted that it is
sizes of the promoter. For stem tissues, significant located most of the time at the same place with respect to
differences were found only between plants with 0.267 kb
the presence of the first intron. Experiments of site directed
and 2.59 kb of promoter size. This result is most likely due
mutagenesis with a PME gene from Aspergillus niger strain
to the few independent transformants used in the analysis.
5344 had shown that there is an histidine residue essential
However, the trend is clear and similar in all plant for PME activity within the amino acid sequence HQAVA
differentiated tissues analyzed.
(Duwe and Khanh, 1996). From Figure 3, we can see that
most of the PME enzymes from higher plants has the
sequence HQAVA as well. This seems to suggest that this
histidine residue can be playing an important role in the
We have cloned and analyzed a genomic DNA region
catalytic activity of the enzyme. Multiple sequence
containing an almost complete and novel PME gene. alignment failed to locate the sequence of HQAVA of
Several tools were used to probe that this region encodes
Erwinia chrysanthemi PMEA or PMEB at the same
the genomic sequence of a PME gene. Comparison of the
location as plant PME’s. However, pair comparison
sequences of PMEU1 genomic coding region with the
between PMEU1 and PMEA or PMEB from Erwinia
PMEU1 cDNA already cloned showed that both are chrysanthemi correctly aligned the sequence HQAVA at
identical with the exception of the intron sequences located
the same position.
in the genomic clone. Further, analysis of the cDNA
sequence using BLAST resulted in high similarity with
Studies of the three-dimensional structure of Erwinia
several DNA regions encoding PME genes. Also, chrysanthemi pectin methylesterase (PME-A) support the
transgenic plant overexpressing the PMEU1 cDNA under
presence of two aspartate and one arginine residues in the
the control of the cauliflower mosaic virus showed higher
active site of the enzime (Jenkins et al. 2001) and not an
levels of PME activity as compared with control plants. It
histidine. However, some of the PME isoenzymes show an
was also shown that this high level of PME activity aspartate residue instead of histidine in the same site
correlated with the presence of a band hybridizing with a
PMEU1 specific probe (Gaffe et al. 1997).
We believe that the study of the possible involvement of
The PMEU1 gene is presented in the tomato genome as a
either an histidine or an aspartate residues in the catalytic
single copy (Gaffe et al. 1997), in contrast with other PME
activity of PME from higher plants deserves further
genes published which had been shown to form clusters
(Richard et al. 1996; Turner et al. 1996).
Computer analysis of the PMEU1 genomic region showed
We perform several experiments to find another copy of the
that this sequence follows several features commonly
gene, like increasing the number of plaques screened and
present in other genes from higher eukaryotic organisms, as
using probes from the 5´ end of the gene with unsuccessful
mentioned above. The phylogenetic analysis (Figure 4) had
results. Also, DNA blot analysis of the 8.4 kb of the 3´ end
shown that this PME gene is not related with other PME
of the DNA inserted in the phage did not show any genes isolated from the tomato genome (Harriman et al.
hybridization with PMEU1 probe even under low 1991). Rather, from Figure 4, we can see that PMEU1 is
stringency conditions (data not shown). Further, DNA blot
more related to two genes from Arabidopsis thaliana
analysis of the tomato genome using EcoR I as restriction
(AtPME2 and AtPME3) and two genes from Citrus sinensis
enzyme showed one band hybridizing to a 6.0 kb band,
(PECS-1.1 and PECS-1.2). Efforts to find a correlation
which correspond precisely with the fragment released from
between relatedness of the PME genes and pattern of
the DNA phage and shown to hybridize with the PMEU1
expression were not succesful. However, the finding just
specific probe (data not shown). Taken together, these
mentioned further support that the cloned PME gene
evidences support that the PMEU1 gene is presented as a
described in this work belong to a entirely novel type of
single copy in the tomato genome and that it is part of the
PME gene from tomato.
DNA contained by the isolated phage from the genomic
Experiments carried out in our lab with tobacco transgenic
Comparison of the PMEU1 genomic coding region with the
plants overexpressing the PMEU1 gene and tomato plant
PMEU1 cDNA sequence showed the presence of two with lower levels of this gene did not produce a change in
introns with 106 and 1039 bp in size (Figure 2). We
the plant phenotype that could be give us an insight as to
compared the structure of genomic regions encoding PME
what is the physiological role of the PMEU1 gene.
genes in regard to the number and size of introns. The
Therefore, we decided to computer analyzed the PMEU1
analysis did not show any clear pattern of structure since
promoter sequence to look for DNA boxes or elements with
there is a high variability in both the size and the number of
known function, in search for insights as to what can be the
introns present. However, when we compared the amino
physiological role of this PMEU1 gene.
acid sequence of 23 PME genes from higher plants and a
PME gene from E. chrysanthemi (Figure 3), the analysis
In Figure 5, it is presented the sequence of the DNA
highlighted a large region in common for most of the plant
regulatory region of the PMEU1 gene. We are not sure of
Isolation and study of a ubiquitously expressed tomato pectin methylesterase regulatory region
having the complete genomic sequence of the PMEU1 gene
by reducing the size of the promoter, its transcriptional
for two reasons: the DNA segment of the PMEU1 gene was
activity is also reduced. As can be seen, even 267 bp of the
located toward the 5´ end of the tomato genomic DNA
PMEU1 regulatory region is transcriptionally active. This
carried by the isolated phage (Figure 1). Further, computer
means that we did not reach the lower limit where the
analysis of the PMEU1 promoter 5´ end region failed to
promoter loose completely its transcriptional activity,
find elements known to exist toward the 3´ end of the gene
although a large reduction was accomplished. In contrast, it
coding regions. However, considering the size of the largest
was reported that a truncated piece of 440 bp of a flax PME
sequence of a PME regulatory region published to date, 2.3
promoter (Lupme3) lost completely the ability to drive
kb (Albani et al. 1991), it is quite possible that we almost
transcription of a reporter gene (Roger et al. 2001). The
had the entire PMEU1 regulatory region. Our efforts to
results of GUS activity in leaf tissue are supported by the
isolate from the tomato genomic library the remaining
histochemical staining analysis in which the transgenic
segment of the PMEU1 regulatory region were largely
plants showed weaker activity in the parenchyma tissue
surrounding the leaf vascular tissue with decrease in the
promoter size (data not shown).
The computer analysis of the PMEU1 regulatory region
showed the presence of both direct repeats and perfect
The change in transcriptional activity among the different
inverted repeats. In Figure 5, only the largest ones are
sizes of promoter is of 6 fold when comparing the 0.267 kb.
shown. It is interesting that repeats 1 and 2, which are only
with the 1.306 kb. and 4 fold when comparing the 1.306 kb.
separated by one base pairs appears to come from only one
with the 2.59 kb. There is a difference of 1.03 kb between
repeat in which a mutation took place, splitting this long
0.267 kb and 1.306 kb and 1.29 kb between 1.306 kb and
repeats into two shorter ones. Also, some of the largest
2.59 kb. The differences in sizes are similar and still the
perfect inverted repeats are present inside of the largest
variation in activity is higher between the 0.267 Kb. and
direct repeats. It can be interested to test whether this
1.306 kb which means that perhaps there are stronger
repeats belong to the PMEU1 promoter or they are part of
enhancer element(s) in the promoter region closest to the
the intergenic region of the plant genome which is known
ATG. Overall, we obtained up to 95% in reduction of
to contain repeat sequences. However, the possible role if
PMEU1 promoter transcriptional activity with the construct
any of these repeats remains to be elucidated.
including 0.267 kb of PMEU1 promoter. Reduction of the
promoter size which brings an associated reduction in
We also located two sequences identical to cis-acting promoter activity as measured with a reporter gene had
elements found in the phytochrome A3 promoter (Bruce et
been found in deletion studies of other promoters (Darasiela
al. 1991). Also, it showed two more sequences similar to
et al. 1996; Royo et al. 1996), however, sometimes smaller
known cis-acting elements regulated by light. From here,
pieces are able to drive higher levels of reporter gene
the possible regulation of this PMEU1 gene by light activity in general (Canevascini et al. 1996) or at some
deserves further attention. We also located a sequence
specific tissues (Royo et al. 1996).
similar to a known abscisic acid responsive element, close
to the transcription start site (Figure 5). The phytohormone
The standard deviation of the parameter is indicating a very
ABA had been related to the abscission phenomena en
high variability which is most likely due to the presence of
plants (Label et al. 1994; Aneju et al. 1999) and to the plant
multiple copies in the genome of the different transformants
responses to abiotic stress in plant (Zhu, 2001). One of the
(not determined), dissimilarities in the physiological status
genes encoding a pectin methylesterase isolated from Citrus
among the leaf tissues used and to the position effect
sinenis was shown to be up-regulated in abscission zones of
(Wilson et al. 1990). This result is alike with studies
leaves (Nairn et al. 1998). Currently, experiments in our
reported earlier, in which a high variability among
laboratory are being carried out to test the possible role of
independent transformants was also found in liquid cell
the gene PMEU1 in the plant responses to light, abscission
cultures expressing the GUS gene under the manopine
and abiotic stress, however, a possible function for the
synthase (Peach and Velten, 1991). Also, tobacco cells
PMEU1 gene in these phenomena is still matter of stably transformed with a chimeric construct in which the
CaMV35S was driving the expression of GUS, showed a
standard deviation three times higher than average for the
With the goal to demonstrate that the 5´ flanking region of
GUS specific activity parameter (Allen et al. 1993).
the PMEU1 genomic clone correspond with an active
regulatory region, and to find the smallest size of the region
The average of GUS activity in tobacco leaf for the
able to direct transcription, we created transgenic tobacco
construct harboring the 2.59 kb of promoter size was
plants expressing different constructs in which the uidA
324.334 pMoles of MU/min/mg protein (Figure 7). This
gene, encoding the enzyme ?-glucuronidase, is being activity is similar to the one reported earlier for tobacco
regulated by different regions of the PMEU1 promoter.
(Nicotiana tabacum var Samsun) leaf of about the same
size used in this work, harboring GUS (uidA gene) under
In Figure 6, it is shown the results of analyzing the ?-
the control of the cauliflower mosaic virus 35S promoter:
glucuronidase activity of around 50 independent 321 pMoles/min/mg protein (Jefferson et al. 1987). This
transformed tobacco plants. From the figure, it is clear that
result suggest that PMEU1 promoter is as strong as the
Tiznado, M. E. et al.
CaMV35S which in turn indicates its usefulness in ANEJU, M.; GIANFAGNA, T. and NG, E. The roles of
overexpressing proteins in plants.
abscisic acid and ethylene in the abscission and senescence
of cocoa flowers. Plant Growth Regulation, March 1999,
We also studied the expression of the three constructs in the
vol. 27, no. 3, p. 149-155.
three main plant tissues: root, stem and leaf of six
independent transformants (Figure 7). It is clear from the
AUSUBEL, F.M.; BRENT, R.; KINGSTON, R.E.;
graph that the three constructs showed the same pattern
MOORE, D.D.; SEIDMAN, J.G.; SMITH, J.A. and
already observed for leaf tissue. However, we recorded 1.7
STRUHL, K. Current Protocolos in Molecular Biology:
and 8 fold PMEU1 transcriptional activity for stem and
Screening of Recombinant DNA Libraries. John Wiley &
root, respectively. This suggest a difference in the strength
Sons, Inc. 1988. p. 1600. ISBN 047150338X.
of the enhancer elements present in the PMEU1 promoter
depending upon the type of plant tissue. These results also
BARRAS, F.; van GIJSEGEM, F. and CHATTERJEE,
suggest that the enhancer element(s) are active en several
A.K. Extracellular enzymes and pathogenesis of soft rot
differentiated tissues and are not specific for leaf tissue.
Erwinia. Annual Review of Phytopathology, September
These results are supported by the GUS histochemical
1994, vol. 32, p. 201-234.
staining in which the transgenic plants harboring the
construct including the smallest promoter region showed
BEVAN, M. Binary vectors for plant transformation.
weaker activity in the parenchyma tissue surrounding the
Nucleic Acids Research, November 1984, vol. 12, no. 22, p.
vascular tissue as compared with tissues of transgenic 8711-8721.
plants expressing the construct with the highest promoter
region (data not shown).
BRADFORD, M. A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing the
These findings are in contrast with deletion studies of other
principle of protein-dye binding. Analytical Biochemistry,
promoter in which it was found that for specific tissues,
May 1976, vol. 72, p. 248-254.
smaller pieces of the regulatory regions are able to direct
higher values of reporter gene enzymatic activity (Royo et
BREWBAKER, J.L. and KWACK, B.H. The essential role
of calcium ion in pollen germination and pollen tube
growth. American Journal of Botany, 1963, vol. 50, p. 859-
Deletion studies of the PMEU1 promoter could be of 865.
significant insight to locate this putative enhancer elements.
However, stronger experimental evidences are needed to
BRUCE, W.B.; DENG, X.W and QUAIL, P.H. A
probe their presence in the PMEU1 promoter region.
negatively acting DNA sequence element mediates
phytochrome-directed repression of phyA gene
In summary, we had isolated an entirely new gene encoding
transcription. EMBO Journal, October 1991, vol. 10, no.
a pectin methylesterase isozyme from the tomato genome
10, p. 3015-3024.
which is represented by a single copy. It shows an
ubiquitous pattern of expression, in contrast with the tissue
CANEVASCINI, S.; CADERAS, D.; MANDEL, T.;
specific gene isolated earlier from tomato. Analysis of its
FLEMING, A.J.; DUPUIS, I. and KUHLEMEIER, C.
promoter region suggest several potential function for this
Tissue-specific expression and promoter analysis of the
gene and we believe that further analysis of this gene will
tobacco ltp1 gene. Plant Physiology, October 1996, vol.
bring new insights to understand better the physiological
112, no. 2, p. 513-524.
role of the pectin methylesterase enzyme.
CHAMBERLAND, H.; OUELLETTE, G.B.; PAUZE, F.J.
and CHAREST, P.M. Immunocytochemical localization of
tomato pectinesterase in root cells of tomato plants infected
ALBANI, D.; ALTOSAAR, I.; ARNISON, D.G. and by Fusarium oxysporum f. sp. radicis-lycopersici.
FABIJANSKI, S.F. A gene showing sequence similarity to
Canadian Journal of Botany, June 1991, vol. 69, no. 6, p.
pectin esterase is specifically expressed in developing 1265-1274.
pollen of Brassica napus. Sequences in its 5´ Flanking
Region are Conserved in Other Pollen-Specific Promoters.
CHEN, M.H. and CITOVSKY, V. Systemic movement of
Plant Molecular Biology, April 1991, vol. 16, no. 4, p. 501-
tobamovirus requires host cell pectin methylesterase. Plant
Journal, August 2003, vol. 35, no. 3, p. 386-392.
ALLEN, G.C.; HALL, G.E.; CHILDS, L.C.; CHRISTGAU, S.; KOFOD, L.V.; HALKIER, T.;
WEISSINGER, A.K.; SPIKER, S. and THOMPSON, W.F.
ANDERSEN, L.N.; HOCKAUF, M.; DORREICH, K.;
Scaffold attachment regions increase reporter gene DALBOGE, H. and KAUPPINEN, S. Pectin methyl
expression in stably transformed plant cells. Plant Cell,
esterase from Aspergillus aculeatus: expression, cloning in
June 1993, vol. 5, no. 6, p. 603-613.
yeast and characterization of the recombinant enzyme.
Isolation and study of a ubiquitously expressed tomato pectin methylesterase regulatory region
Biochemical Journal, November 1996, vol. 319, no. 3, p.
Carolina, USA). Plant Physiology, Rockville, MD, USA. p.
COLLMER, A. and KEEN, N.T. The role of pectic GREEN, P.J.; KAY, S.A. and CHUA, N.H. Sequence-
enzymes in plant pathogenesis. Annual Review of specific interaction of a pea nuclear factor with light-
Phytopathology, September 1986, vol. 24, p. 383-409.
responsive elements upstream of the rbcS-3A gene. EMBO
Journal, September 1987, vol. 6, no. 9, p. 2543-2549.
DARASELIA, N.D.; TARCHEVSKAYA, S. and
NARITA, J.O. The promoter for tomato 3-hydroxy-3-
GUILTINAN, M.J.; MARCOTTE, Jr., W.R. and
methyl-glutaryl coenzyme a reductase gene 2 has unusual
QUATRANO, R.S. A plant leucine zipper protein that
regulatory elements that directs high-level expression. Plant
recognizes an abscisic acid response element. Science,
Physiology, October 1996, vol. 112, no. 2, p. 727-733.
October 1990, vol. 250, no. 4978, p. 267-271.
DITTA, G. A n