Planta (1991)185:38~45

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~l-lt~

9 Springer-Verlag1991

Cloning of a cDNA encoding 1-aminocyclopropane-l-carboxylate synthase and expression of its mRNA in ripening apple fruit Jian Guo Dong 1, Woo Taek Kim ~, Wing Kin Yip 1, Gregory A. Thompson 2, Liming Li 1, Alan B. Bennett ~, and Shang Fa Yang 1 * 1 Mann Laboratory, Department of Vegetable Crops, Universityof California/Davis,and 2 Calgene Inc., 1920 Fifth Street, Davis, CA 95616, USA Received 5 February; accepted 22 March 1991

Abstract. l-Aminocyclopropane-l-carboxylate (ACC) synthase (EC 4.4.1.14) purified from apple (Malus sylvestris Mill.) fruit was subjected to trypsin digestion. Following separation by reversed-phase high-pressure liquid chromatography, ten tryptic peptides were sequenced. Based on the sequences of three tryptic peptides, three sets of mixed oligonucleotide probes were synthesized and used to screen a plasmid cDNA library prepared from poly(A) + RNA of ripe apple fruit. A 1.5-kb (kilobase) cDNA clone which hybridized to all three probes were isolated. The clone contained an open reading frame of 1 214 base pairs (bp) encoding a sequence of 404 amino acids. While the polyadenine tail at the 3'-end was intact, it lacked a portion of sequence at the 5'-end. Using the RNA-based polymerase chain reaction, an additional sequence of 148 bp was obtained at the 5'-end. Thus, 1 362 bp were sequenced and they encode 454 amino acids. The deduced amino-acid sequence contained peptide sequences corresponding to all ten tryptic fragments, confirming the identity of the eDNA clone. Comparison of the deduced amino-acid sequence between ACC synthase from apple fruit and those from tomato (Lycopersicon esculentum Mill.) and winter squash (Cucurbita maxima Duch.) fruits demonstrated the presence of seven highly conserved regions, including the previously identified region for the active site. The size of the translation product of ACC-synthase mRNA was similar to that of the mature protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), indicating that apple ACC-synthase undergoes only minor, if any, post-translational proteolytic processing. Analysis of ACC-synthase mRNA by in-vitro translation-immunoprecipitation, and by Northern blotting indicates that the ACC-synthase mRNA * To whom correspondenceshould be addressed Abbreviations: ACC = 1-aminocyclopropane-l-carboxylic acid; AdoMet= S-adenosyl-L-methionine;HPLC= high-pressure liquid chromatography; kDa=kilodalton; kb=kilobase; mAb=monoclonal antibody; Met = methionine; PCR = polymerasechain reaction; poly(A)+ RNA = polyadenylatedRNA; SDS-PAGE= sodium dodecylsulfate-polyacrylamidegel electrophoresis

was undetectable in unripe fruit, but was accumulated massively during the ripening proccess. These data demonstrate that the expression of the ACC-synthase gene is developmentally regulated.

Key words: 1-Amninocyclopropane-l-carboxylate synthase - eDNA - Ethylene synthesis - Fruit ripening Gene expression - Malus

Introduction Ethylene is a plant hormone which regulates many processes of plant growth, development and senescence (Abeles 1973). The biosynthesis of ethylene in higher plants has been elucidated to proceed by the following pathway (Adams and Yang 1979): methionine (Met)~Sadenosyl-L-methionine (AdoMet) ~ 1-aminocyclopropane- 1-carboxylic acid (ACC)--* C z H 4. Ethylene production in plant tissues is normally low but is greatly promoted at certain developmental stages such as fruit ripening, by auxin application, or under certain environmental stresses (Yang and Hoffman 1984). In these cases, increased ethylene production results from increased ACC synthesis from AdoMet catalyzed by ACC synthase (EC 4.4.1.14). Thus, ACC synthase has been the major subject of investigation concerning the regulation of ethylene biosynthesis. Because of its low abundance, progress in the purification of ACC synthase has been slow. The enzyme was purified and immunologically characterized in tomato (Bleecker et al. 1986; Satoh and Yang 1988; Van Der Straeten et al. 1989), winter-squash (Nakagawa et al. 1988; Nakajima et al. 1988), apple (Dong et al. 1991 ; Yip et al. 1991) and zucchini-squash (Sato and Theologis 1989) fruits. During the final stage of our work, two papers appeared which described the isolation and sequencing of ACC-synthase cDNA clones from ripe and wounded tomato fruit (Van Der Straeten et al. 1990), and wounded winter-squash fruit (Nakajima etal. 1990) tissues. In both tissues, ACC-synthase mRNA was enriched by mechanical wounding.

J.G. Dong et al. : ACC-synthase eDNA of apple O u r interest has focused o n the expression o f A C C synthase d u r i n g d e v e l o p m e n t a l l y regulated fruit ripening. We have isolated A C C synthase f r o m ripe apple fruit (Yip et al. 1991) a n d raised m o n o c l o n a l a n t i b o d i e s ( m A b ) a g a i n s t it ( D o n g et al. 1991). Partially purified apple A C C synthase was r a d i o l a b e l e d with Ado[14C]M e t or N a B 3 H , a n d purified b y i m m u n o a f f i n i t y chrom a t o g r a p h y . F o l l o w i n g t r y p s i n digestion, the r a d i o a c tive active-site p e p t i d e was sequenced (Yip et al. 1990). I n this paper, we r e p o r t the i s o l a t i o n o f a r i p e n i n g - r e g u lated A C C - s y n t h a s e e D N A clone f r o m apple fruit f r o m a e D N A library u s i n g m i x e d o l i g o n u c l e o t i d e p r o b e s corr e s p o n d i n g to sequences o f tryptic peptides o f apple A C C synthase. We have sequenced the e D N A a n d characterized the expression o f the A C C - s y n t h a s e gene in r i p e n i n g apple fruit.

Material

and methods

Plant material. Apple (Malus sylvestris Mill., cv. Golden Delicious) fruit was harvested from a local orchard. The internal ethylene concentration of individual fruit was measured by withdrawing a 100-gl gas sample from the core tissue of the fruit. The fruit was classified into three ripening stages according to the internal ethylene concentration and the fruit-skin color: stage I (preclimacteric) fruit was green with an internal ethylene concentration below 0.2 gl/l; stage II (climacteric rise) fruit was green and had internal ethylene between 5 and 20 gl/l; and stage III (climacteric) fruit was yellowish green and had ethylene above 200 gl/1. The fruit was stored frozen at - 8 0 ~ C until needed.

39 to a nylon membrane (Zeta-Probe; BioRad, Laboratories, Richmond, Cal., USA). The filter was hybridized to azP-labeled cDNA probe as described by Kim and Okita (1988). The filter was washed twice with 1 x SSPE (250 mM sodium chloride-10 mM sodium phosphate (pH 7.6)-1 mM EDTA) containing 0.1% SDS for 15 rain at room temperature, twice with 0.2 x SSPE for 15 min at room temperature, and finally twice for 30 rain in 0.2 x SSPE at 65~ C. The blots were visualized by autoradiography at - 8 0 ~ using Kodak XAR-5 film Eastman-Kodak, (Rochester, N.Y. USA) and an intensifying screen (Crones; Dupont, Wilmington, Dela., USA).

Production of a eDNA and colony screening. A eDNA library was constructed in the cloning vector pCGN 1703 from poly(A) + RNA isolated from ripe apple fruit by the method of Alexander (1987) with the modifications described by O'Neill (1989). A eDNA reaction with 2 gg of vector and 5 gg of poly(A) + RNA yielded 105 independent transformants. The plasmid library was screened by colony hybridization (Hanahan and Meselson 1980) using one of the three oligonucleotide probes listed in Fig. 1. After hybridization to one probe, the radioactive hybrids were removed by boiling the filter, and hybridized to the next probe. Subcloning and sequence analysis, eDNA inserts were excised from pCGN by Xba I or Pst I digestion, separated by gel electrophoresis and recovered by electro-elution. The inserts were ligated into Bluescript SK plasmid (Stratagene, La Jolla, Cal., USA). Doublestranded template DNA was prepared according to Sambrook et al. (1989). The 5'- and 3'-ends of each clone were sequenced using Reverse and T7 primers according to the Sequenase sequencing kit manual (US Biochemical, Cleveland, Ohio, USA). The above three sets of oligonueleotide probes were used as sequencing primers to obtain three internal sequences. Based on the three inter-

Partial purification of ACC synthase and production of monoclonal antibody. Detailed procedures for the partial purification of ACC

Tryptic Peptides 1:

NPEAAAFK

synthase (Yip et al. 1991) and its mAb (monoclonal antibody) production (Dong et al. 1991) have been reported. A mAb designated as 6A10 was used in this study for Western blotting, immunoprecipitation and immunoaffinitypurification.

2:

YPGFDR

3:

ALEEAYOEAEK

4: 5:

NATFNSHGEDSSY VGAIYSNDDMVVAAATK

Immunoaffinity purification, trypsin digestion and amino-acid sequencing of tryptic fragments. Immunopurifieation, trypsin digestion and amino-acid sequencing of tryptic fragments were carried out as previously described (Yip et al. 1990). Briefly, mAb 6A10 was immobilized to protein A-agarose and used to isolate ACC synthase from a partially purified ACC-synthase preparation. The purified protein was subjected to trypsin digestion, and the resulting tryptic peptides were separated by reverse-phase HPLC (highpresure liquid chromatography). Each fraction was further purified by a second dimentional HPLC separation, and sequenced.

Isolation and gel blot hybridization of RNA. Total RNA was prepared from frozen apple-fruit mesocarp tissues at various ripening stages. The RNA was isolated as described by Lay-Yee et al. (1990) with slight modifications. One hundred grams of frozen tissue were homogenized in 200 ml of lysis buffer containing 4.0 M guanidine isothiocyanate, 10mM EDTA (dithylenediamine tetraacetate), 300raM Tris (2-amino-2-(hydroxymethyl)-l,3-propanediol, pH 7.5, 10% (v/v)2-mercaptoethanol, 0.5% n=lauroylsarcosine and 0.5% Nonidet P-40 (Sigma, Chemical Co., St. Louis, Mo., USA). Total RNA was pelleted through a cesium chloride gradient by centrifugation at 25000 rpm with a Beckman (Fullerton, Cal., USA) SW 28 rotor for 40 h. The pellet was resuspended in 10 mM Tris (pH 7.6), 1 mM EDTA, 0.1% SDS (sodium dodecyl sulfate) and 500 mM NaC1, Polyadenylated mRNA was purified by two rounds of oligo(dT)-cellulose chromatography. Five micrograms of poly(A) + RNA isolated as above was separated by electrophoresis in a 1.2% agarose gel containing formaldehyde, and blotted

6:

SNTFEAEMELWK

7:

AFVGEYYNVPEVN

8: 9:

AMV - FMAEIR GVLVTNPSNPLGTITITR

10: SLSKDLGLPGFR Oligonucleotide Probes 1:

5'-GAA-GAA-GCI-TAC-CAA-GAA-CGI-GAA-AA-3' G G T G G G

2:

5'-AAC-ACI-TIC-GAA-GCI-GAA-ATG-GA-3'

3:

5'-AAA-GAC-CTI-GGI-CTA-CCI-GG-3'

T G

T TT

G

G C G

T Fig. 1. Amino-acid sequences of ten tryptic peptides derived from purified apple ACC synthase and nucleotide sequences of three tryptic oligonucleotide probes prepared on the basis of the peptide sequences. Single-letter code is used for amino-acid sequence from amino to carboxyl terminus; -indicates that the amino-acid residue could not be identified. Except for peptide 3, all other peptides were derived from a tryptic digest and are listed in order of HPLC elution. Underlined sections of the peptide sequences indicate regions used for the design of oligonucleotide probes. Probes 1, 2 and 3 were based on peptides 2, 6, and 10, respectively. I stands for the inosine substitute

40

J.G. Dong et al. : ACC-synthase cDNA of apple

Xhol AJnalll

I

Aflll

II

Narl EcoRI Hindlll

Sacl Snal

Sacll

I !1

II

I

111.3 1 1 2 8

1388

I

1,36 1 7 5

427

744

874 820

Apal

I

I 1636

1525

TI~A~T~ACAcGGGGAAGACTCCTCcTAcTT~TTAGGTTGGcAAGAGTATGAGAAGAAcCc~TAccATGAGGT~cTCAACAcAAA~GGGATTATTcAG F

N

S

H

G

E

D

S

S

Y

F

L

G

W Q

E

Y

E

K

N

P

Y

H

E

V

k

N

T

N

G

I

l

Q

ATGGGTCTAGCAGAAAATCAGCTCTGTTTTGATCTTCTCGAGTcATGGcTGGCTAAGAATC~AG~GCAGcTGCATTTAA~AAAATGGAGAATcCATA198 M G L A E N Q L C F D L L E S W L A K N P E A A A F K K N G E S I TTTGCAGAGCTTGcTCTcTTCCAAGATTATcATGGcCTTcCCGcGTTCAA~AGGcAATGGTAGATTTCATGGCG~AATTCGAGGG~CAAAGTGACC 297 F A E L A L F Q D Y H G L P A F K K A N V D F N A K/E I R G N K V T TTTGATcCC~CCACTTAGTGCT~ACCGCcGGTGCAACTTCAGCGAATGAGAcCTTTATTTTcTGCCTTGcTGAcCccGG~GAAGCCGTTCTTATTccT 396 F D P N H L V L T A G A T S A N E T F I F C L A D P G E A V L I P AcCcCATAcTA~CCAGGATTTGATAGAGACcTTAAGTGGCGAAcTGGAGTCGAGATTGTAC~CATTcACTGcAc~GCTCC~TGGCTTcCAAATTACT 495 T P Y Y P G F D R D L K W R T G V E I V P I H C T S S N G F 0 I T GAAA~CGCTCTGG~GAAGCCTACC~GAAGCCGAAAAACGCAATCT~AGAGTCAAAGGAGTcTTGGTcA~G~CC~ATCAAACcCATTGGGCACCACA 594 E T A L E E A Y Q E A E K R N L R V K G V L V T N P S N P L G T T ATGACCAGAAACGAA~TCTA~CTc~TCcTTTCCTTCGTTGAAGACAAGGGCATCcACcTCATTAGCGATGA~TTTACTC~GGcACAGcTTTTAGcTcc 693 H T R N E L Y L L L S F V E D K G I H L I S D E I Y S G T A F S S CCAT~cTTTAT~GCGTcATGG~GTT~TCAAAGATAGGAACTGTGATGAGAATTccG~GTTTGGcAGCGAGTTCAcGTTGT~TATAGCCTcTCTAAG 792 P S F I S V H E V L K D R N C D E N S E V W Q R V H V V Y S L S K GATCTTGGCCTTC~GGGTTTTcGAGTTGGCGCCATcTA~TccAACGACGAcATGGTTGTGGCCGCCGCTAc~AAATGTCAAGCTTTGGTcTTGTTTCT 891 D L G L P G F R V G A I Y S N D D H V V A A A T K M S S F G L V S TCTCAAACTCAG•AcCTTcTCTCcGcCATGcTATCCGACAAAAAAcTCACTAAGAAcTACATAG•CGAGAA•cACAAAAGACTCAAACAACGTCAGAAA 990 S 0 T Q H L L S A M L S D K K L T K N Y I A E N H K R L K Q R Q K AAGCTcGTCTcCGG•CTTCAGAAATCTGGCATTAGcTGCCT•AACGGCAATGcTGGcTTGTTcTGTTGGGTGGATATGAGGCA•TTGCTTAGGTCCAA• K L V S G L Q K S G I S C L N G N A G L F C W V D N R H L L R S N

1089

ACCTTTGAAGCCGAAATGGAGcTCTGGAAAAAGATTGTATAcGAAGTT•A•cTCAATATATCTCCTGGATCGTCTTGTCATTGCACGGAACCTGGTTGG 1188 T F E A E N E L W K K I V Y E V H L N l S P G S S C H C T E P G W TTCcGTGTcTG~TTTGCC~CTTGcCCGAGAGAAcTCTGGACTTGGCAATGcAGAGA~TGAAGGCATTTGTGGGGGAGTATTACAACGT~CCTGAGGTC1287 F R V C F A N L P E R T L D L A H Q R L K A F V G E Y Y N V P E V AATGGCGGCAAGcAGCCATTTAAGCCACTCAAGAAGACAGTCGCT•AcAAAGTGGGTTTCCCGGCTATCCTTCGATGAc•GCGGTcCTATT•cCGGTAG N G G K Q P F K P L K K T V A H K V G F P A I L R

13~

ATGAAAGGTAGCCTGGTCTGAGTA~GA~CCGCT~GGAAAATTACATTTTAGACCAAACATTTTTTcTGCCAAAAAGTTAATTGGTTGAATTTTTT 1485 TTTCGTTTTAGTTTTTTTTTTTT•TCCGAATGTAGAGAAGTGcACACGGTCcGTGTTTGGGGATGTGAAGTGCGTTTcGCTTcATTTGTAAAAAGGGTG 1584 TGTTATCCACATACCCTATTTGACTTCTCA~AAAAAAAA~AAAA~AA

Fig. 2. DNA sequence and its deduced amino-acid sequence of apple ACC synthase. The pAAS2 nucleotide sequence is shown in nucleotides 149-1636, while PCR amplification of ACC-synthase m R N A yielded a sequence corresponding to nucleotides 1 338.

The two sequences overlap precisely in a region of 190 bp (nucleotides 149-338). Amino-acid sequences identical to those ~ u n d in the tryptic peptides are u n ~ r ~ e ~ Partial restriction map of the clone is shown on the t ~

nal sequences obtained, additional oligonucleotides (20-mers) were synthesized and used directly as primers for further sequencing.

sis, and the remainder was diluted tenfold with phosphate-buffered saline containing 1% Nonidet P-40 and 1% bovine serum albumin (BSA). The reaction mixture was first incubated with pre-immune serum and precipitated with protein A-Sepharose (Sigma). The resulting supernatant was then incubated with mAb 6A10 followed by protein A-Sepharose precipitation. Immunoprecipitation was carried out as previously described (Dong et al. 1991). Total translation products and the solubilized immunoprecipitate were separated by SDS-PAGE and visualized by fluorography.

RNA-based polymerase chain reaction (PCR). Total RNA isolated by cesium-chloride gradient centrifugation as described above was purified by phenol-chloroform extraction followed by precipitation with ethanol twice. The first-strand cDNA was synthesized by mouse leukemia-virus reverse transcriptase (Kawasaki et al. 1988), directed by a gene-specific primer (nucleotides 674-693; Fig. 2). The P e R was carried out in a total volume of 20 HI containing 5 ~tl of the first-strand rection products, 1 HM primers, 50 mM KCI, 10 mM Tris (pH 8), 1.5 mM MgClz, 0.01% gelatin, 200 ~tM deoxynucleotides, and 2.5 units of Taq polymerase (Perkin Elmer/ Cetus, Norwalk, Conn., USA). The reaction was performed for 30 cycles at 94 ~ C denaturation (I min), 50~ C annealing (2 min), and 72 ~ C extension (2 min) in an automatic Thermal Cycler (Perkin-Elmer Cetus). The P e R products were sequenced as described above after subcloning into Bluescript plasmid. In-vitro translation and immunoprecipitation. One microgram poly(A) + RNA or 60 I-tg total RNA, isolated from ripe apple-fruit mesocarp, was translated in vitro with rabbit reticulocyte lysate according to the manufactor's instruction (Boehringer Mannheim Biochemicals, Indianapolis, Ind., USA) in the presence of [35S]Met (Amersham Corp., Arlington Heights, Ill., USA; 37 TBq/mmol, 3.7 MBq/reaction). A 5-pJ aliquot was used for SDS-PAGE analy-

Electrophoresis and immunoblotting. Protein samples, total translation products and immunoprecipitated translation products were separated by SDS-PAGE using 12% acrylamide according to Laemmli (1970). Electrophoretic blotting and detection methods were as described previously (Dong et al. 1991). Biotinylated protein molecular standards were prepared according to Della Penna et al. (1986). DNA hybridization. Apple genomic DNA was isolated from leaf tissue by cesium chloride gradient centrifugation as described previously (Kim and Okita 1988), digested with appropriate enzymes, and separated by electrophoresis in a 0.6% agrose gel. The gels were blotted to nylon filters (Zeta-Probe), prebybridized and hybridized to 32p-labeled cDNA probe. After hybridization, the filter was washed and subjected to autoradiograpby as described for RNA gel blotting.

J.G. Dong et al. : ACC-synthase cDNA of apple

41

Results

Amino-acid sequencing of tryptic peptides. Immunoaffinity-purified ACC-synthase protein was subjected to trypsin digestion followed by reversed-phase H P L C separation. Ten well-separated major fragments were se-

Fig. 3. Comparison of molecular size between the in-vitro translation product and the mature protein of apple ACC synthase. Poly(A) + RNA isolated from ripe apple fruit was in-vitro-translated with rabbit reticulocyte lysate. The total translation product was immunoprecipitated with mAb 6A10, and the resulting immunoprecipitate was subjected to SDS-PAGE analysis. A partially purified ACC-synthase preparation (450 units with a specific activity of 9500 units/mg protein) was similarly separated on the same gel. The proteins of both samples on the gel were transferred to a nitrocellulose filter, which was immunoblotted with mAb 6A10 and analyzed by fluorography. Lane 1, fluorogram of the in-vitro translation products followed by immunoprecipitation with mAb 6A10; lane 2, immunoblot analysis of a partially purified ACCsynthase preparation detected with mAb 6A10

350 d.

300

Screening and identification of ACC-synthase eDNA clones. Primary screening o f about 200000 colonies with the oligonucleotide probes described above yielded one positive clone which hybridized to all three probes. This putative ACC-synthase clone, pAAS1, had a c D N A insert of only 1.3 kb, which is not long enough to encode the 48-kDa mature form o f ACC synthase (Dong et al. 1991). In order to isolate a longer c D N A clone, an oligonucleotide complementary to the 5'-end o f the pAAS1 clone (nucleotides 274-293; Fig. 2) was used as a probe to re-screen the library. Three clones were isolated. Sequencing analysis showed that one o f the clones was identical to pAAS1 and all three clones contained overlapping regions and 3'-end poly(A) + tails. The longest clone, designated as pAAS2, had a 1.5-kb insert (nucleotides 149-1636; Fig. 2). The insert has one open reading frame encoding 404 amino-acid residues. This reading frame did not begin with the initiation codon A T G , but ended with a poly-A sequence. Since the deduced aminoacid sequence contained sequences corresponding precisely to the 10 tryptic peptides, we concluded that the c D N A clone encoded ACC synthase but lacked the 5'end of the sequence. Extension of uncloned cDNA sequence. In order to extend the uncloned portion o f the c D N A clone, RNA-based PCR was carried out. When the purified ACC-synthase protein prior to tryptic digestion was subjected to Nterminal sequencing, a mixed 17-amino-acid sequence was obtained. Although the first 6 cycles (S/M-V/L-M/ K - L / E - V / F - K / R ) were ambiguous, the subsequent 11 cycles contained a sequence (N-A-T-F-N-S-H-G-E-D-S) which overlapped with one of the 10 tryptic peptides, peptide 4 (Fig. 1). We reasoned that this tryptic peptide must be located near the N-terminus o f the ACCsynthase protein. Therefore, an RNA-based P C R was designed employing a 20-mer oligonucleotide complementary to nucleotides 319-338 (Fig. 2) as the downstream primer and mixed oligonucleotide sequence

35

m Ethylene I ~ ACC synthase

30

i

250

25

-~ c-

200

20

"$

150

15

100

10

|

quenced (Fig. 1). From these sequences, three partial sequences (underlined) were chosen to construct corresponding degenerate oligonucleotide probes shown in Fig. 1.

~n

E

C

0

E c

--

5

50

0

0

II Stages of ripeness

III

t-

o c t9

Fig. 4. Changes in internal ethylene concentration and ACC-synthase activity of apple fruit at various ripening stages. Stages L / / a n d Ill represent the preclimacteric, climacteric-rise and climacteric-peak stages

42

J.G. Dong et al. : ACC-synthase cDNA of apple

Fig. 5A, B. In-vitro translation of RNA isolated from apple at different stages of ripeness. About 60 lag of total RNA isolated from apple fruit at stages I (lane 1), II (lane 2) and III (lane 3) were in-vitro-translated (A) and the translation products were immunoprecipitated with mAb 6A10 (B). These products were analyzed by SDS-PAGE and fluorography. Lance C represents total translation products in the absence of apple RNA

[TT(C/T)AA(C/T)TC(A/T/C/G)CA(C/T)GG(A/C/G/T)G A ( A / G ) G A ] deduced from peptide 4 as the upstream primer. A P C R product of 338 bp was specifically amplified (nucleotides 1-338; Fig. 2). Two P C R (products (PCR1, PCR2) were obtained from two independent amplifications. Sequence analysis of PCR1 indicated that the 190 bp at the 3'-end overlapped precisely with p A A S 2 at its 5'-end. Thus, an additional sequence of 148 bp (nucleotides 1-148; Fig. 2) was obtained at the 5'-end. It is to be noted that the deduced amino-acid sequence f r o m this extended sequence contained the sequences o f two tryptic peptides (peptides 1 and 4, Fig. 1) derived f r o m a purified protein. These nucleotide-sequence results fully confirm that the tryptic peptide of N - A - T - F - N - S - H - G - E - D - S - S - Y was indeed located near the N-terminus. Sequencing of P C R 2 showed that it was identical to PCR1 except that nucleotide 274 was G instead of A. Hence, the deduced amino-acid residue at position 92 was K in both PCR1 and pAAS2, but was E in PCR2. This discrepancy m a y result from sequence heterogeneity in apple A C C synthase or from a nucleotide and-or amino-acid sequencing error. Since the nucleotide A-274 was found in all three c D N A clones the sequence corresponding to PCR1 was chosen for the alignment of the extended sequence. In-vitro translation of ACC-synthase mRNA. In apple extracts, ACC-synthase activity is associated with a 48k D a protein (Dong et al. 1991 ; Yip et al. 1991). In order to determine if apple A C C synthase is processed to the mature form by post-translational modifications, we c o m p a r e d the molecular size of the primary translation

Fig. 6. Northern blot analysis of apple-fruit poly(A) + RNA. Poly(A) + RNA was obtained from ripening apple fruit at stages I (lane 1) and III (lane 2), resolved by formaldehyde-agarose gel electrophoresis, and blotted onto a membrane filter. The blot was then probed with 32p-labeled pAAS2 cDNA clone, washed, and subjected to autoradiography Fig. 7. Genomic Southern blot analysis of the apple ACC-synthase gene. DNA (10 lag) prepared from apple leaf tissue was digested with either PstI (lane P), BamHI (lane B), or XbaI (lane JO and resolved on a 0.6% agarose gel. DNA on the gel was transferred to a nylon filter, probed with 32p-labeled pAAS2 cDNA clone, washed, and subjected to autoradiography

product and the mature ACC-synthase protein. Polyadenylated R N A isolated from ripe apple fruit was translated in vitro using rabbit reticulocyte lysate, and the products subjected to immunoprecipitation with m A b 6A10 (Dong et al. 1991). A partially purified ACCsynthase preparation (450 units with a specific activity of 9500 units/mg protein) was loaded adjacent to the immunoprecipitated translation product, and simultaneously separated by SDS-PAGE. Following fluorography, a major translation product of 48 k D a and a minor polypeptide of about 40 k D a were detected. It is not known whether this minor translation product resulted from an unrelated antigen, from proteolysis of A C C synthase, or from aberrant synthesis in a heterologous system (reticulocyte lysate). The major translation product (48 kDa) had a similar mobility to the purified ACCsynthase protein visualized by immunoblotting (Fig. 3). These results indicate that the native protein and the polypeptide encoded by ACC-synthase m R N A have the same molecular size.

J.G. Dong et al. : ACC-synthase cDNA of apple

43 1

APPLE TOMATO SQUASH

(xxxxxxNAT)FNSHGEDSSYFLGWQEYEKNPYHEVLNTNGIIIQMGLAENQL M G F E I - - A K T N S I L S K L * * N E E * * * N * P * * D * * K A * D S D * F * P L K * P * * V ~********** M E F H Q I D E R N Q A L L S K I * V D D G * * * N * P * * D * * K A * D N D * F * P E D * P L * V I********** 2 CFDLLESWLAKNPEAAAFKKNGESIFAELAL FQDYHGLP AFKKAMVDFMAKIRGNKVTFDPNHLVLTAGATSANET *L**I*D*IKR**KGSICS-E*IKS*KAI*N ******** *********************************** S**MIVD*IR*H***SICTPK*LER*KSI*N ******** E*RNGIAS**G*V**GR*Q***SRI*MGG***G*S**

41

"117

4

F IFCLADPG E A V L I P T P Y Y P G F D R D L K W R T G V E I V P I H C T S S N G F Q I T E T A L E E A Y Q E A E K R N L R V K G V L V I T N P S N 193 I ******** * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * V ******** **********AA*********RA****************KA******KK************* ***** 5 PLGT--~ M T R N E L Y L L L S F V E D K G I H L I S D E I Y S G T A F S S P S F I S V M E V L K D R N - - - C D E N S E V W Q R V H V V l Y S L S K D L 266 *****I L D K D T * K S V * * * T N Q H N * * * V C * * * * A A * V * D T * Q * V * I A * I * D E Q E M T Y * N K D L ..... **I*I******M *****I Y D * D T * K T * V T * * N Q H D * * * * C * * * * * A * V * K A * T * * * I A Q I V E - - E M E H * K K E L ..... I * I L I * * * * * * M 6

GLPGFRVGIAIYSNDDMVVAAATK ************************ ***********************

MSSFGLVSISQTQHLLSAMLSDKKLTKNYIAENHKRLKQRQKKLVSGLQKSGIS *************************************************** ***************************************--**********

CLNGNAGLFCWVDMRHLLRSNTFEAEMELWKKIVYEVHLNISPGSSCHCTIEPGWFRVCFAN *************************************************************** *************************************************************** F V G E Y Y N V P E V N G G K Q P F K P L K K T V A H K V G F P A I LR * * * - V - E K S G D K S S S M E K * Q Q W * K N N L R L S * S - - K * M Y D E S - V L S P L S SP I P P S P L V R * *ENI-DKK*D*TVAM* S*TRRRENKLRLS* SFSG*RYDEGNVLNSPHTMSPHSPLVIAKN Fig. 8. Comparison of deduced amino-acid sequences of ACC syn-

thases from apple, tomato (Van Der Straeten et al. 1990) and winter squash (Nakajima et al. 1990) fruits. Amino-acid residues are numbered based on the apple amino-acid sequence starting with the residue F. Sequence identity and similarity among the three species are 43% and 83%, respectively. *, Amino-acid residues in tomato or winter-squash enzyme that are identical to apple ACC synthase;

Expression of the ACC-synthase gene. To study the expression o f the ACC-synthase gene during fruit ripening, two experiments were carried out. In the first experiment, we analyzed the translatable ACC-synthase m R N A at three different ripening stages. At stage I (preclimacteric stage), ethylene concentration was as low as 0.17 gl/1 and ACC-synthase activity was undetectable. At the onset o f ripening (stage II), both internal ethylene and ACC-synthase activity increased substantially. At a late ripening stage (stage III), internal ethylene concentration exceeded 300 gl/1 and ACC-synthase activity was about 40 units/mg protein (Fig. 4). Total R N A was isolated from apple fruit at each stage and subjected to in-vitro translation and immunoprecipitation (Fig. 5). The 48-kDa in-vitro translation product was not observed in unripe (stage I) fruit, but was detected in stage II fruit and maximally in stage III fruit. We have analyzed poly(A) + R N A isolated from apple fruit at the preclimacteric (stage I) and late ripening stage (stage III) by R N A blot hybridization using the pAAS2 c D N A clone as a probe. Figure 6 shows that the m R N A is about 2.0 kb in size and is detectable only in ripening apple fruit tissues. Even with prolonged exposure of the film, no signal was detected in preclimacteric fruit. Together, these data indicate that increased ethylene and ACC-synthase activity in ripening apple is closely correlated with increased level of ACC-synthase m R N A .

I LPERTLDLAMQRLKA MDDG*V*I*LA*IRR MDDN*V*V*LN*IHS

342

418

454

-, sequence gap. Based on the N-terminal and tryptic peptide sequences, there are a total of nine amino-acid residues at the Nterminus of apple ACC synthase, which are not deduced from the DNA sequence (Fig. 2). These nine residues are represented inside parentheses; among them the six N-terminal residues are unidentified and are represented by x, whereas the last three residues are predicted from peptide sequence to be N-A-T

Genomic Southern analysis. Apple genomic D N A was digested with BamHI, XbaI or PstI, neither of which cleaved the ACC-synthase c D N A insert, and these digests were probed with 3ZP-labeled pAAS2. In the XbaI and BamHI digests, three fragments of D N A with different hybridization intensities were observed, while in the PstI digest only one band was detected (Fig. 7). Discussion The identification o f pAAS2 as encoding apple ACC synthase is supported by the observation that the aminoacid sequences of tryptic peptides obtained from purified ACC-synthase protein were present in its deduced amino-acid sequence (Fig. 2). Moreover, the deduced amino-acid sequence of this apple c D N A shows high homology to tomato and winter-squash cDNAs. The nucleotide sequences of all three c D N A clones isolated from apple fruit contained a termination codon and polyadenylation tails at the 3'-end but were missing some sequence at 5'-ends. Using PCR amplification an additional sequence of 148 bp at the 5'-end was obtained. Probably because our ACC-synthase preparation was not sufficiently pure, N-terminal sequencing yielded a mixed 17-amino-acid sequence. Since peptide 4 (Fig. 1) had the sequence of N-A-T-F-N-S-H-G-E-D-S which was found

44 in cycles 7-17 of the N-terminal amino-acid sequence, we assume that nine amino-acid residues are still absent from the 5'-end coding region o f our nucleotide sequence (Fig. 2). O f the nine amino-acid residues missing at the N-terminus, the last three residues are predicted to be N-A-T, based on the tryptic-peptide amino-acid sequence. Taken together, ACC synthase in apple fruit is predicted to contain at least 463 amino-acid residues, 454 of which were deduced from our c D N A (Fig. 2). Attempts to obtain the 5'-end sequence using Poly(A) + RNA-based primer extension (Zimmern and Kaesherg 1978) and P C R techniques (Loh et al. 1989) have been unsuccessful. The failure of these experiments may be a consequence o f the low aboundance of ACC-synthase m R N A in the poly(A) + R N A preparation. Three ACCsynthase c D N A clones were obtained by screening about 4. l0 s recombinant clones, indicating that the abundance of ACC synthase m R N A was about 0.001% of total m R N A . The protein level of the apple enzyme was estimated to be about 0.0021% (Dong et al. 1991 ; Yip et al. 1991). Furthermore, isolation of poly(A) + R N A might be another limiting factor. We have only succeded in isolating R N A from apple fruit by cesium-chloride gradient centrifugation. Efforts are being undertaking to obtain the 5'-end sequence information by constructing a new c D N A library and-or genomic library. The primary translation product of ACC-synthase m R N A had approximately the same molecular size as its mature protein (Fig. 3), indicating that ACC synthase in ripe apple fruit undergoes only minor, if any, posttranslational proteolytic processing. This is different from ACC synthase in wounded tomato (Van Der Straeten et al. 1990) and winter-squash (Nakajima et al. 1990) fruit. In tomato, it was proposed that an 85-amino-acid sequence might be eliminated at the C-terminus of the protein. In winter squash it was reported that ACC synthase was converted from a 58-kDa to a 50-kDa form resulting in a loss of about 70 amino-acid residues at the C-terminus (Nakajima et al. 1988). The hydropathy profiles of tomato and winter-squash ACC synthases showed a large number o f hydrophilic groups at the C-terminus. However, the hydropathy profile of the amino-acid sequence deduced from our apple c D N A does not indicate similar properties at the C-terminus. It should be noted that both tomato and winter-squash ACC synthases were induced by mechanical wounding, while the apple enzyme was isolated from ripening fruit. The deduced amino-acid sequences of ACC synthase from apple, tomato and winter-squash fruits show a high degree of conservation (Fig. 8), with a 52%, 53% and 62% sequence identity between apple and tomato enzymes, apple and winter-squash enzymes, and tomato and winter-squash enzymes. Although the amino-acidsequence identity among all three species is only 43%, the overall similarity is as high as 83%. There are seven highly conserved regions, designated as regions 1 to 7, which contain at least eight amino-acid residues and show greater than 95% identity (Fig. 8, boxed regions). We have previously identified region 5 as the active-site center, where K-264 is responsible for binding its coenzyme pyridoxal 5'-phosphate and catalyzing the enzy-

J.G. Dong et al. : ACC-synthase cDNA of apple matic reaction (Yip et al. 1990). We have isolated and sequenced the active-site peptide of both apple and tomato enzymes. While residue L-266 was found in the active-site peptide of apple enzyme, both methionine and leucine were found at this position in tomato enzyme. We have postulated that there are at least two ACCsynthase isoenzymes in tomato fruit, one with leucine and the other with methionine in the active-site peptide. Besides the seven conserved regions, it is of interest that there is a divergent region near the C-terminus (residues 425-454). Since these amino-acid sequences are not conserved, one could speculate that this region of ACC synthases may not be directly related to enzyme activity. The accumulation of ACC-synthase m R N A as detected by in-vitro translation and by Northern blotting was closely correlated with fruit ripening. These data indicate that the c D N A clone we have isolated is the product of a ripening-related gene. In tomato, two different ACC-synthase c D N A clones were isolated from a library which was constructed from m R N A isolated from wounded and ripe tomato fruit (Van Der Straeten et al. 1990; Olson et al. 1991). In winter squash it was demonstrated that the wound-induced ACC synthase was immunologically different from the IAA-induced enzyme (Nakagawa et al. 1988). Thus, it is reasonable to assume that ACC synthase is encoded by a multi-gene family, and that each gene is separately activated by a specific internal or external factor, such as fruit ripening, IAA or mechanical wounding. Cloning of the ACCsynthase c D N A represents the first step toward understanding the control of ethylene biosynthesis at the molecular level. This work was supported by grants DCB-9004129 and INT8915155 from the National Science Foundation.

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