©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Analysis of LE-ACS3, a 1-Aminocyclopropane-1-carboxylic Acid Synthase Gene Expressed during Flooding in the Roots of Tomato Plants (*)

David C. Olson (§) , Jürg H. Oetiker (¶) , Shang Fa Yang

From the (1) Mann Laboratory, Department of Vegetable Crops, University of California, Davis, California 95616

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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)()(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.

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(11) . However, the molecular basis of ACC synthase induction, including identification of the exact gene(s) induced by anaerobiosis, was not determined.

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.


MATERIALS AND METHODS

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) .


RESULTS

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.

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.


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.

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) 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) .


DISCUSSION

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) 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.

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.


FOOTNOTES

*
This work was supported by U. S. Dept. of Agriculture-National Research Initiative Competitive Grants Program Grant 9203120 and National Science Foundation Grant MCB-9303801. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) M38822 (sequence of pBTAS3, revised) and L34171 (sequence of LE-ACS3).

§
To whom correspondence and reprint requests should be addressed. Tel.: 916-752-0714; Fax: 916-752-4554; E-mail: dcolson@ucdavis.edu.

Supported by a Swiss National Science Foundation fellowship.

The abbreviations used are: ACC, 1-aminocyclopropane-1-carboxylic acid; LE, Lycopersicon esculentum; ACS, ACC synthase; PCR, polymerase chain reaction; kb, kilobase(s); bp, base pair(s).

Since publication, the sequence of pBTAS3 has been revised and deposited in the GenBank data base, accession number M38822.

The sequence of LE-ACS3 has been deposited in the GenBank data base, accession number L34171.


ACKNOWLEDGEMENTS

We would like to thank James Lee for technical assistance and Dr. Rick Baker for helpful discussions on intron splicing.


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