tomato (LEACC2G) and apple (pAASZ), respectively (Dong ... of the broccoli, tomato, and apple clones were aligned ... Dr. E. Almira, Interdisciplinary Center.
Plant Physiol. (1995) 108: 857-858
Plant Gene Register
Nucleotide Sequence of a cDNA Clone Encoding 1-Aminocyclopropane-1-Carboxylic Acid Synthase- from BroccoIiBarry J. Pogson', Christopher C. Downs, Kevin M. Davies*, and Stephen C. Morris Division of Horticulture, Commonwealth Scientific and Industrial Organization, P.O. Box 52, North Ryde 21 13, New South Wales, Australia (B.J.P., S.C.M.); and New Zealand lnstitute for Crop & Food Research Ltd., Private Bag 4005, Levin, New Zealand (C.G.D., K.M.D.) Ethylene regulates many processes in plant growth, development, and senescence. Two enzymes, ACC synthase and ACC oxidase, are responsible for ethylene biosynthesis from its precursor S-adenosyl Met (Yang and Hoffman, 1984), and blocking the synthesis of these enzymes in antisense RNA experiments results in reduced ethylene synthesis (Hamilton et al., 1990; Oeller et al., 1991). Functional identification of cDNA clones encoding ACC synthase has been achieved by expression studies in Escherichia coli and yeast and by sequence comparisons to peptides purified from ACC synthase protein (Sato and Theologis, 1989; Yip et al., 1990; Dong et al., 1991). Broccoli florets are composed of both vegetative and floral tissues that senesce rapidly after harvest. The most visible change is Chl degradation in the sepals, which begins within 48 h of harvest (tissues held in darkness at ZO'C), and is associated with increases in ACC oxidase mRNA and ethylene synthesis from within the florets (Tian et al., 1994; B. J. Pogson, unpublished data). We report the sequence of a cDNA clone (pbroc3, Table I) isolated from a cDNA library in AZapII (Stratagene) constructed from poly(A+) A " from broccoli florets held at 2OoC in darkness for 48 h after harvest. The cDNA is 1734 bp long and encodes an open reading frame of 481 amino acids. The nucleotide and predicted amino acid sequences show high identity with ACC synthase from tomato (LEACC2G) and apple (pAASZ), respectively (Dong et al., 1991; Rottmann et al., 1991). T h e predicted peptides of the broccoli, tomato, and apple clones were aligned (Devereux et al., 1984), and regions of high identity were found to correspond to conserved regions of other ACC synthases (Park et al., 1992). The pbroc3 cDNA contained a region with 83% amino acid identity (100% similarity based on conservative changes) with the peptide sequence identified as the active site of ACC synthase (Yip et al., 1990).
Table I. Characterization o f pbroc3 cDNA from senescing florets of broccoli Organism: Broccoli (Brassica oleraceae L. var ltalica cv Shogun). Techniques: Double-stranded plasmid sequencing by chain termination method using synthetic oligonucleotides as primers. Method of Identification: A c D N A library from florets held 48 h after harvest (at 20°C in darkness) was screened with an apple cDNA clone (pAAS2; Dong et al., 1991). Sequence comparison showed a high degree of nucleotide sequence identity with functionally identified ACC synthase clones, pAAS2 and LEACC2G from apple (54%) and tomato (64%),respectively (Dong et al., 1991 ; Rottmann et al., 1991). The predicted amino acid sequence shows 52 and 64% identity with apple and tomato, respectively, and has very high identity with conserved regions of other ACC synthases, including a region with 83% amino acid identity (100% similarity based on conserative changes) with the peptide identified as the active site of ACC synthase (Yip et al., 1990; Park et al., 1992). Features of the Deduced Protein: The open reading frame is 491 amino acids with a predicted M, of 55,000; the protein i s neither strongly hydrophillic nor hydrophobic; the estimated p l is 5.9. The subcellular localization of the protein has not been determined. There were 57 b p upstream of the start Met. The 3 ' untranslated region was 204 bp long and included a putative polyadenylation signal and site. Expression Characteristics: Transcript of approximately 1700 nucleotides; the transcript abundance did not change in florets at O, 24, 48, and 72 h after harvest and was detected in similar abundance in green and senescing leaves. Ant ibodies: None available. Gene Copy Number: Not determined.
ACKNOWLEDCEMENTS W e are grateful to Shang Fa Yang for providing the c D N A clone pAAS2 used in this study. Dr. E. Almira, Interdisciplinary Center
for Biotechnology Research, University of Florida, provided a commercial sequencing service.
'
Received November 15, 1994; accepted December 21, 1994. Copyright Clearance Center: 0032-0889/95/108/0857/02. The EMBL accession number for the sequence reported in this article is X82273.
Present address: Department of Plant Sciences, University o f Arizona, Tucson, AZ 85721. * Corresponding author; e-mail daviskQcrop.cri.ng; fax 64-63683578. 857
Pogson et al.
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Plant Physiol. Vol. 108, 1995
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