From the
The plant hormone ethylene is produced in response to a variety
of environmental stresses. Previous work has shown that flooding or
anaerobic stress in the roots of tomato plants caused an increase in
the production of the ethylene precursor
1-aminocyclopropane-1-carboxylate (ACC) in the roots, due to
flooding-induced activity of ACC synthase (EC 4.4.1.14). RNA was
extracted from roots and leaves of tomato plants flooded over a period
of 48 h. Blot analysis of these RNAs hybridized with probes for four
different ACC synthases revealed that the ACC synthase gene LE-ACS3 is rapidly induced in roots. LE-ACS2 is also induced, but
at later times. The genomic clone for LE-ACS3 was isolated and
sequenced. At all time points, the probe from the LE-ACS3 coding region hybridized to two bands in the RNA blots.
Hybridization using the first and third introns of LE-ACS3 separately as probes indicate that flooding may inhibit processing
of the LE-ACS3 transcript. Sequence homology analysis
identified three putative cis-acting response elements in the
promoter region, corresponding to the anaerobic response element from
the maize adh1 promoter, the root-specific expression element
from the cauliflower mosaic virus 35S promoter and a recognition
element for chloroplast DNA binding factor I from the maize chloroplast
ATP synthase promoter.
The plant hormone ethylene can be induced by a variety of
factors including fruit ripening, senescence, auxin application,
wounding, and numerous biotic and abiotic stresses
(1) . The
enzyme ACC synthase (S-adenosyl-L-methionine
methylthioadenosine-lyase, EC 4.4.1.14), catalyzes what is recognized
as the rate-limiting step of ethylene biosynthesis, the conversion of
S-adenosyl-L-methionine to
1-aminocyclopropane-1-carboxylate
(ACC)
The
physiological response of plants to one specific abiotic stress,
flooding, has been well characterized. Waterlogging of the roots of
tomato (Lycopersicon esculentum) results in epinasty of
petioles, leaf chlorosis and senescence, reduced stem elongation, and
adventitious root formation
(3, 4) . Since it was shown
that flooding causes an increase in ethylene evolution
(5) , and
that flooding symptoms are similar to those caused by
ethylene
(4, 6) , it was suggested that ethylene may be
responsible for the flooding damage symptoms
(7, 8) .
Later studies in which an anaerobic environment was created around the
root demonstrated that the root was the source of ethylene
production
(9) . Bradford and Yang
(10) subsequently
clarified the biochemical and physiological pathway of flooding-induced
ethylene production. Under conditions of low oxygen tension caused by
flooding, roots are stimulated to synthesize ACC, presumably through
increased ACC synthase activity. The ACC is then transported via the
xylem to the shoot and leaves where it is rapidly converted to ethylene
by ACC oxidase. That ACC synthase activity is induced by anaerobiosis
has been confirmed by finding an increase in extractable ACC synthase
activity in tomato roots flushed with N
The identification of the specific ACC synthase gene(s)
that is flooding-induced is important to better understand and possibly
ameliorate the deleterious effects of flooding. In tomato, the DNA
sequences of four different ACC synthase genes have been described.
These clones have been described as PCR fragments or full-length
genomic or cDNA clones. The gene LE-ACS2 has been shown to be
induced by fruit ripening, wounding, auxin, treatment with fungal
elicitors or Phytophthora
infestans(12, 13, 14, 15, 16) .
LE-ACS4 is induced by fruit
ripening
(12, 13, 14, 15) . LE-ACS3 and LE-ACS5 are induced by auxin and
wounding
(14) . In this laboratory, four PCR fragments, pBTAS1,
-2, -3, and -4, were isolated as probes for the genes LE-ACS2,
-3, -5 and -4, respectively
(14) .
These fragments all correspond to an approximately 300-bp portion of
the coding region which encodes the area surrounding the active site of
the enzyme. The relative DNA sequence identity of these probes ranges
from 52% to 60%, except for pBTAS1 and pBTAS4 which have a 72%
identity.
In this paper we identify the LE-ACS3 and
LE-ACS2 genes as being induced in tomato roots in response to
flooding and describe the time course of their induction. We also
describe the isolation of the LE-ACS3 genomic clone, the
processing of its transcript, and the location of possible
cis-acting regulatory elements of its promoter.
In order to
induce flooding stress in the root, 12-week-old tomato plants were
submerged to the cotyledonary node for up to 48 h in nutrient solution.
Plants were then tested at intervals over the entire 48 h of flooding
for ethylene production by the leaf tissue. In a typical experiment,
ethylene evolution measured from leaves taken from the flooded plants
began to increase at 24 h and increased 15- to 20-fold, relative to the
nonflooded plants, at 48 h. These results are consistent with previous
results demonstrating an induction of ACC production by
flooding
(10, 11) . Therefore, we then analyzed the
transcriptional induction of specific ACC synthase genes.
In order
to determine which ACC synthase gene was being induced, RNA was
extracted from roots which had been flooded for 0, 1, 2, 4, 10, 24, and
48 h. When RNA blots were hybridized with each of the four ACC synthase
probes, the probes which correspond to LE-ACS3 and LE-ACS2 demonstrated an induced signal in roots (Fig. 1). While the
signal intensity of each time point varied somewhat among several
different experiments, the LE-ACS3 transcript was consistently
present after only 1 h of flooding and remained present throughout the
times analyzed, increasing in intensity at the 24-48-h time
points. The LE-ACS2 transcript began to accumulate only at 10
h, peaking at 24 h, then declining at 48 h. The other two probes showed
no detectable hybridization to these RNAs, suggesting that LE-ACS4 and -5 are not induced by flooding. Since some ACC
synthase activity may be present in the leaves (11), RNA was also
extracted from leaves at 0-, 10-, and 48-h time points. No
hybridization to any of the four probes was ever detected in leaf RNA
(data not shown). Therefore, any transcripts from the four ACC
synthases corresponding to our probes are either not present in leaves
or are beyond the detection limits of our methods.
To ascertain whether both bands
hybridizing to the LE-ACS3 probe represent polyadenylated
transcripts, as well as to determine the induction pattern of all four
ACC synthases at higher sensitivity, poly(A)
The response of tomatoes to flooding has been well
characterized with regard to the induction of ethylene synthesis.
Previous physiological data demonstrated that after a period of root
anaerobiosis, ACC synthase is induced in roots, resulting in the
increased synthesis and accumulation of ACC and that this ACC was
transported to the leaves where ACC exerts its physiological effect
through conversion to ethylene
(10, 11) . This report
establishes certain molecular events which precede the production of
ACC in flooded roots: the rapid transcriptional induction of the
LE-ACS3 gene, as well as the LE-ACS2 gene at later
times.
The induction of root-specific LE-ACS3 transcription
is rapid, within 1 h, and is accompanied by a later rise in ethylene
evolution by the leaves. Since no ACC synthase transcript was
detectable in the leaf tissue, we infer that the increased ethylene
evolution in the leaves results not from an increased synthesis of ACC
in the leaves but from ACC transported from the flooded root. Since the
increases in ACC synthase activity
(11) and ACC
production
(11, 34) are preceded by an accumulation of
LE-ACS3 transcript, it is reasonable to conclude that the
active ACC synthase in the root may be LE-ACS3. However, the
LE-ACS2 gene is also induced, albeit not as quickly, but still
at times which coincide with the increase in ethylene production, and
hence may contribute to the increase in ethylene synthesis. Whether
these two are the only ACC synthase genes induced remains open to
further investigation. In addition to the four ACC synthases tested,
amino acid sequences from two other ACC synthases have been
reported
(13) , and other undiscovered ACC synthase genes may
also be expressed in flooded tissues. Furthermore, we have detected
only an increase in the level of transcript and not in enzyme activity
specific to any ACC synthase gene, so conclusive proof will await
isolation of monoclonal antibodies specific for individual ACC synthase
genes, or construction of transgenic plants expressing an antisense
copy of the specific gene.
Expression studies of flooding-induced
LE-ACS3 always revealed the presence of two RNA bands
hybridizing to a coding region probe. The two bands showed the same
pattern of induction over time and are not due to cross-hybridization
to ribosomal RNA. Hybridization of poly(A)
The presence of both the
processed and unprocessed forms of the LE-ACS3 transcript is
intriguing, but stress-induced incomplete splicing of transcripts is
not uncommon. The hsp70 gene of petunia
(35) and the
gmhsp26A gene of soybean
(36) both display disrupted
splicing when exposed to heavy metals like cadmium or copper. Another
gene, adh1 of maize, accumulates up to 10% unspliced RNA
during hypoxia
(37) . These cases all involve stress-induced
splicing failure, which could also be the mechanism operating in
LE-ACS3 during flooding. However, not all splicing failure is
due to stress. The Bronze-2 gene of maize shows unspliced
transcript in purple husk tissue. This splicing failure is influenced
both by genetic and physiological factors
(38) . The presence of
both the processed and unprocessed forms of the LE-ACS3 RNA
may reflect an aspect of some sort of post-transcriptional
regulation
(39) . Since ACC synthase transcripts may be unspliced
under different inducing stimuli, it remains to be seen whether
splicing represents a general control mechanism for ethylene
production.
Comparison of the sequence of the promoter region with
known cis-acting regulatory elements revealed similarity with
three characterized response elements. While the presence of a sequence
matching the chloroplast DNA binding factor (CDF1) site may be
coincidental, sequences identical with two other elements fit well in
the context of the LE-ACS3 expression pattern in the flooded
root. A pentamer found twice in the cauliflower mosaic virus as-1
element, shown to be necessary for root-specific expression, is found
in the LE-ACS3 promoter. This element may control the
LE-ACS3 expression in the root. A sequence identical with a
core consensus element from subregion II of the maize adh1 anaerobic response element (ARE), a region shown to be necessary
for anaerobic induction, is also present in the LE-ACS3 promoter, although inverted on the complementary strand. Since
this subregion of the ARE has been shown to function in either
orientation (27), the fact that its orientation in LE-ACS3 is
reversed may not be important. While the position of the root-specific
response element in the LE-ACS3 promoter is similar to its
location in the cauliflower mosaic virus 35S promoter, the anaerobic
response motif is much further upstream in the LE-ACS3 promoter relative to its position in the adh1 promoter.
Further analysis is needed to determine whether these elements actually
function in regulating LE-ACS3 expression.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank
We would like to thank James Lee for technical
assistance and Dr. Rick Baker for helpful discussions on intron
splicing.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)(1) . The cloning of the gene for
this enzyme has led to a rapid increase in our understanding of its
induction at the molecular level. Individual ACC synthase genes have
been shown to be induced in a tissue-specific manner by ripening,
wounding, and treatment with auxin (for a review, see
Kende
(2) ). However, the expression pattern of these genes in
response to the full complement of ethylene-inducing agents, in
particular certain abiotic stresses, has not been determined.
(11) .
However, the molecular basis of ACC synthase induction, including
identification of the exact gene(s) induced by anaerobiosis, was not
determined.
Growth and Flooding Treatment of Tomato
Plants
Tomato plants (L. esculentum Mill. cv. VFN8)
were grown in pots of 10-cm diameter in a growth chamber at 70%
relative humidity under a photoperiod of 16 h of day at 25 °C and 8
h of dark at 20 °C. Plants were watered with half-strength
Hoagland's solution
(17) . Twelve-week-old plants were
flooded for up to 48 h by submerging the potted plant up to its
cotyledonary node in a tub containing half-strength Hoagland's
solution. Leaf tissue was collected and assayed immediately for
ethylene, and root and leaf tissues were quick-frozen for later RNA
extraction.
Ethylene Determination
Sections of the second or
third youngest mature leaves were used for determining ethylene
production, following the procedure of Jackson and
Campbell
(18) . Excised leaves were capped in a 16-
100-mm tube. After 30 min, 1-ml air samples were removed by a syringe
and injected into a gas chromatograph equipped with a flame ionization
detector for ethylene determination.
RNA Extraction and Hybridization
Root and leaf
tissues were frozen in liquid N, ground to a fine powder,
and extracted by the SDS-phenol method
(19) with minor
modifications. After the phenol-chloroform extractions, the nucleic
acids were precipitated with 1 volume of isopropyl alcohol. The pellet
was resuspended and precipitated twice with 2 M LiCl. Further
purification was needed for some preparations that had high
polysaccharide levels. The RNA was resuspended in water, followed by
addition of one-half volume of ice-cold ethanol, and incubation for 30
min on ice. The polysaccharides were then pelleted by centrifugation at
25,000
g, and the RNA in the supernatant was ethanol
precipitated. Poly(A)
RNA was isolated using the
Poly(A)Ttract system (Promega). For RNA blotting, the RNA samples were
run on 1.2% agarose-formaldehyde gels and blotted to Zeta-Probe
(Bio-Rad) as described
(15) . Hybridizations to
P-labeled probes and subsequent washes followed previously
described methods (15). ACC synthase probes used for hybridizations
were the XbaI-HindIII inserts from pBTAS1, -2, -3,
and -4 (14).
(
)
The probe for the first intron of
LE-ACS3 was generated by PCR using 5`-GTAATTTGAAAAAAATACTTG-3`
as the forward primer and 5`-CTGCGTAACGAAAATAAAAT-3` as the reverse
primer, and the cloned LE-ACS3 gene as template. The probe for
the third intron was conveniently excised as an
RsaI-HinfI fragment from the cloned LE-ACS3 gene.
Screening of Tomato Genomic Library for LE-ACS3
A
library constructed from VFNT cherry tomato genomic DNA cloned into
Charon 35 was kindly provided by Dr. Robert Fisher
(20) .
Plating, plaque lifting, and filter hybridization methods were
essentially as described (19). The probe used in screening was the
P-labeled XbaI-HindIII insert of pBTAS2,
corresponding to the gene LE-ACS3.
Cloning and Sequencing
All DNA manipulations were
performed using standard methods
(21) . PCR was performed using
Taq polymerase according to the manufacturer's
specifications (Perkin-Elmer Cetus). The LE-ACS3 clone
was subcloned by digesting the insert with EcoRI and cloning
each of the resulting four fragments into pBluescript SK
(Stratagene). These subclones were sequenced via the
dideoxynucleotide chain termination method using Sequenase 2.0 (U. S.
Biochemical Corp.) according to the manufacturer's protocol.
Clones were sequenced using a combination of deletions from a fixed end
point
(22) or construction of specific internal primers. DNA and
protein sequence analyses were performed using the PCgene software of
Intelligenetics, Signal Scan
(23) , and Transcription Factor data
base
(24) .
Induction of ACC Synthase in Flooded Tomato
Roots
Past work has provided clear evidence that the ethylene
production rate from leaves taken from flooded plants closely
correlates with the induction of ACC synthase and the accumulation of
ACC in the root during flooding-induced
anaerobiosis
(10, 11) . Hence, we extracted RNA from
roots of flooded plants showing increased ethylene production in the
leaf tissue over the time course of flooding treatment.
Figure 1:
Expression of
LE-ACS2, ACS3, ACS4, and ACS5 in
RNA from flooded tomato roots. Twenty µg of total RNA from roots of
plants flooded for 0, 1, 2, 4, 10, 24, and 48 h was electrophoresed on
a formaldehyde-agarose gel, subjected to blotting, and hybridized with
equal amounts of the P-labeled
XbaI-HindIII fragments of: pBTAS1 corresponding to
LE-ACS2 (A), pBTAS2 corresponding to LE-ACS3 (B), pBTAS3 corresponding to LE-ACS5 (C), and pBTAS4 corresponding to LE-ACS4 (D).
In all
repetitions of the RNA blot analyses, the probe for the LE-ACS3 gene consistently produced a hybridization pattern of two bands of
sizes approximately 2.1 and 1.7 kb. Since the blots used total RNA, one
possibility for the two bands would be cross-hybridization to ribosomal
RNA. This explanation can be ruled out, since the relative signal
intensity differs among the various time points with no signal present
at 0 h, while the stained gel showed uniform amounts of ribosomal RNA
in each lane (data not shown). The two bands do correspond to a size
range observed for other ACC synthase
transcripts
(12, 13, 15) . One of the bands could
represent the LE-ACS3 transcript while the other could be due
to cross-hybridization to the transcript of another ACC synthase.
Alternatively, since both bands display the same pattern of induction,
the upper band could represent an unprocessed form of the LE-ACS3 transcript, with the lower band the processed form. We set out to
confirm this possibility.
RNA was
purified from selected time points and the hybridizations were repeated
with all four probes. When hybridized to the LE-ACS3 probe
pBTAS2 (Fig. 2), the signal intensity with poly(A)
RNA is greater, but the overall induction pattern is the same as
with total RNA. As seen previously with total RNA, no signal was
visible with the LE-ACS4 or LE-ACS5 probes, while the
hybridization pattern with the probe for LE-ACS2 was the same
(data not shown). Since the dual banding pattern of the LE-ACS3 transcript is still seen with poly(A)
RNA, the
transcripts have been processed past the addition of the poly(A) tails.
The determination of the identity of these two bands by utilizing other
LE-ACS3-specific probes had to await isolation and
characterization of the genomic clone of LE-ACS3.
Figure 2:
Expression pattern of LE-ACS3 poly(A) RNA from flooded tomato roots. Two µg
of poly(A)
RNA from roots flooded for 0, 1, 4, and 10
h was electrophoresed on a formaldehyde-agarose gel, blotted, and
hybridized to the
P-labeled XbaI-HindIII
fragment of pBTAS2.
Cloning the LE-ACS3 Gene
To obtain information
concerning the structure of the LE-ACS3 gene, as well as to
study its promoter sequence, we proceeded to isolate its genomic clone.
(The characterization of the LE-ACS2 gene, which was induced
by flooding at later times, has been reported elsewhere
(13) .)
500,000 plaques from a Charon 35 tomato genomic library were
screened with pBTAS2, the LE-ACS3 probe, and three positively
hybridizing clones were selected for further investigation. Restriction
analysis of each of these clones revealed the same pattern for the
approximately 10 kb insert, so one clone,
LE-ACS3-1, was
selected for further analysis. When subjected to restriction with
EcoRI, four fragments of sizes approximately 4, 3, 2, and 1.5
kb were visible and were subcloned into pBluescript
SK
. When subjected to DNA blotting with our
LE-ACS3 probe, the 4-kb fragment was shown to contain the
insert with identity to the probe (data not shown). The entire coding
sequence, as well as regions in the 5`- and 3`-untranslated regions
were sequenced on both strands for a total of 4.8 kb, as shown in
Fig. 3
. The 4-kb fragment contained the entire coding sequence as
well as 1.1 kb of the promoter region. The coding region includes 3
introns whose junctions match the consensus intron/exon borders for
plants
(25) , and the predicted amino acid sequence agrees with
that previously reported for LE-ACS3(13) . The coding
region of 1407 bp encodes a protein of 469 amino acid residues with a
predicted mass of 53 kDa. The LE-ACS3 sequence contains a
single mismatch to the reported sequence of the PCR-generated pBTAS2
fragment
(14) , a change of guanine in pBTAS2 to adenine at
position 3378 in LE-ACS3, resulting in a change of a valine to
isoleucine.
Figure 3:
The organization of the clone and
complete nucleotide sequence of the genomic clone of LE-ACS3.
A, the organization graphic shows the overall organization of
the genomic clone with the location of four exons, represented by
shaded boxes, and three introns represented by lines connecting the four exons. B, the complete nucleotide
sequence of the LE-ACS3 gene including exons, introns, and the
5`- and 3`-untranslated regions. The coding sequence is capitalized and highlighted, while the introns and untranslated
regions are indicated in lowercase. Putative caat box, tataa
box, and polyadenylation sites are underlined in bold.
Putative cis-acting recognition sites in the 5`-untranslated
region are in bold and double-underlined. The region
contained in the pBTAS2 probe is delimited by two inverted
triangles. The derived amino acid sequence is presented in the
one-letter code above the DNA sequence and is numbered separately. The star identifies the stop codon. The seven
invariant residues that are known to bind pyridoxal phosphate coenzyme
in various aspartate aminotransferases and are conserved across all
aspartate aminotransferases and ACC synthase are in bold. The
dodecapeptide which is part of the ACC synthase active site is
double-underlined.
Detection of Transcripts by Probing with
Introns
With the isolation of the LE-ACS3 genomic
clone, we now could proceed to further analyze the two bands observed
in the RNA blots. Based upon the location of the plant consensus
polyadenylation site
(32) in the LE-ACS3 gene and a
transcription start site predicted from the known site of two other
tomato ACC synthase genes, LE-ACS2(13) and
LE-ACS4(33) , the sizes of the LE-ACS3 transcript before and after processing of its introns would be
approximately 2.05 and 1.75 kb, respectively, which coincide closely
with the two bands seen in the RNA blots. If the upper band indeed
represents the LE-ACS3 message with its introns intact, then
hybridization with an intron probe should label only this upper band.
The availability of intron probes allowed us to test this possibility.
Intron 1 (nucleotides 2549 to 2663 in Fig. 3) and intron 3
(nucleotides 3070 to 3164 in Fig. 3) were used as probes to
discern the extent of processing in poly(A) RNA
isolated from roots flooded for 48 h. As seen in Fig. 4, the
probe for intron 3 hybridized only to a single band. Stripping the
filter and reprobing it with the pBTAS2 probe for the LE-ACS3 coding region confirmed that the double bands seen previously are
present on the filter, and that intron 3 hybridized only to the upper
band. Intron 1, however, hybridized to two bands. When stripped and
reprobed with the pBTAS2 probe, it is evident that the two bands with
which intron 1 hybridized are the upper band, as well as a band of
intermediate size. This intermediate size band may represent a
partially processed LE-ACS3 transcript or cross-hybridization
to some unknown transcript. These data demonstrate that the
polyadenylated LE-ACS3 transcript accumulates in both the
processed and unprocessed form during flooding-induced anaerobiosis in
tomato roots.
Figure 4:
Expression of the LE-ACS3 transcript when hybridized with intron probes. One µg of
poly(A) RNA from roots flooded for 48 h was
electrophoresed on a formaldehyde-agarose gel, blotted, and probed with
the
P-labeled intron 1 probe (A) or intron 3
probe (C). B and D represent the filters
A and C, respectively, stripped, and rehybridized
with the
P-labeled XbaI-HindIII fragment
of pBTAS2. Filter A was exposed for 8 days, filter C for 4 days, and
filters B and D for 24 h.
Computer Search for Regulatory Elements in the LE-ACS3
Promoter Sequence
To link the expression characteristics of the
LE-ACS3 gene with its promoter region, the sequence of the
promoter was compared with a data base of known cis-acting
regulatory elements. While some similarity with many elements was found
in the search, exact matches were found for three particular elements.
At position 1151 in Fig. 3is the sequence AAACCAC, whose reverse
complement, GTGGTTT, is identical with the core consensus element from
subregion II of the maize adh1 anaerobic response element
(ARE)
(26, 27, 28) . A second element, GTATTTAG,
is located at position 1538, which, also in the inverse complement,
matches exactly with the octamer CTAAATAC. This octamer is found in
tandem in the promoter region of the maize chloroplast ATP synthase
(atpB3) gene and has been shown to be recognized by the
chloroplast DNA binding factor 1 (CDF1)
(29) . The sequence motif
TGACG at position 2131 in Fig. 3matches a motif found in the
cauliflower mosaic virus 35S promoter
(30) . This pentamer is
found twice in the cauliflower mosaic virus as-1 element, which has
been shown to be responsible for root-specific
expression
(30, 31) .
RNA with
probes corresponding to introns 1 and 3, followed by reprobing with an
exon sequence, shows these bands are polyadenylated forms of
LE-ACS3 before and after intron processing and not
cross-hybridization to a different ACC synthase gene. The estimated
size difference of 0.4 kb between the two bands on the RNA blots
corresponds well to the actual combined size of 319 bp for the three
introns in the gene (see Fig. 3). While both introns hybridized
to the upper band, intron 1 also hybridized to a band of intermediate
size. The presence of a band of intermediate size may indicate that
intron 3 is processed out before intron 1. Thus, intron 3 hybridized to
the larger transcript containing all three introns, while intron 1
hybridized to the larger transcript containing all three introns as
well as to an intermediate transcript lacking intron 3 and possibly
intron 2. This could reflect a mechanism in which the introns are
processed out of a transcript progressively in a 3` to 5` direction. An
alternative explanation for the intermediate band could be that intron
1 may cross-hybridize to an unknown transcript showing a similar
induction pattern. Two bands were also observed for the LE-ACS2 transcript, of approximate sizes 1.8 and 1.3 kb. In this case,
however, the expected sizes of the unprocessed and processed
transcripts would be approximately 2.8 and 1.8 kb, based upon the known
transcript size combined with 1064 bp for the three
introns
(13) . Furthermore, in a previous study of ACC synthase
expression, two bands corresponding to the expected size of a processed
transcript and a smaller transcript for the LE-ACS2 gene were
observed in RNA from leaves treated with P. infestans (16).
The exact identity of these two forms of the LE-ACS2 transcript remains to be determined.
/EMBL Data Bank with accession number(s) M38822
(sequence of pBTAS3, revised) and L34171 (sequence of
LE-ACS3).
data base, accession number M38822.
data base, accession number
L34171.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.