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This is to certify that the
dissertation entitled
positive and negative feedback

regulation of ethylene biosynthesis
induced by indole-B-acetic acid

presented by

Scott Charles Peck

has been accepted towards fulfillment
of the requirements for

Ph . D . degree in BQta__1LY____

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MSU le An Afflrrnetive Action/Equal Opportunity lnetitwon

 

POSITIVE AND NEGATIVE FEEDBACK REGULATION
OF ETHYLENE BIOSYNTHESIS INDUCED
BY INDOLE—3-ACETIC ACID
By

Scott Charles Peck

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Botany and Plant Pathology

1995

ABSTRACT

POSITIVE AND NEGATIVE FEEDBACK REGULATION
OF ETHYLENE BIOSYNTHESIS INDUCED
BY INDOLE-3-ACETIC ACID
By

Scott Charles Peck

A cDNA clone, pPE8, encoding l-aminocyclopropane-l-carboxylate (ACC)
oxidase from etiolated pea stems was isolated. The insert was expressed in transgenic
yeast to confirm that it encoded a functional ACC oxidase. The localization of an ACC
oxidase from tomato was studied in transformed yeast. Transgenic ACC oxidase
accumulated in the yeast cells but could not be detected in the vacuole as determined by
activity assays and by immunoblot analysis using ACC oxidase-specific antibodies.
These results indicated that ACC oxidase was not associated with the vacuole as had been
previously suggested. In 5- to @day-old etiolated pea stems, ethylene induced an
increase in ACC oxidase transcript level and enzyme activity. Indole—3—acetic acid
(IAA), which stimulates ethylene production, also caused an increase in ACC oxidase
mRNA levels and enzyme activity. This increase was blocked by 2,5—norbomadiene
(NBD), a competitive inhibitor of ethylene action. An increase in the level of ACC
synthase mRN A and enzyme activity preceded the increase in ACC oxidase levels after
IAA treatment. These results indicate that the enzymes of ethylene biosynthesis are
sequentially induced after treatment of intact pea seedlings with IAA and that ethylene

promotes the accumulation of ACC oxidase mRNA and enzyme activity through a

positive feedback loop. Two cDNA clones of IAA-induced ACC synthase, PS-ACSl and
PS-ACSZ, were isolated. Ethylene inhibited the IAA-induced accumulation of transcripts
of both clones. Results from genomic southern blot analysis, RNA blot analysis using
a probe from the 3’-untranslated region, and oligonucleotide-directed RNAse H mapping
indicated that a single gene for PS—ACSl produces Mo transcripts (1.6 kb and 1.9 kb)

with differences in the 5’-ends of the transcripts.

For my parents

iv

ACKNOWLEDGMENTS

I would like to thank the members of the motion picture academy for voting for - wait,
wrong acknowledgment. I would like to thank the people who have contributed, both
directly and indirectly, to the completion of this thesis. First, I thank Hans Kende for
his support, guidance, and insight over the years; und dass er mich Antje vorgestellt hat -
danke schOn. I thank the members of my committee, Ken Poff, Jon Walton, and Jan
Zeevaart, for their advice and for being merciful during prelims. Though not on my
committee, Pam Green also provided numerous suggestions which greatly aided my
research. Finally, I thank the members of the Kende lab, both past and present, and the
numerous grad students and post-docs who have given both assistance and friendship.
In particular, I raise a big Dale Cooper cup-o-coffee to David Olson for having the
patience to try to teach this liberal arts graduate some molecular biology.

And because sanity must be protected in one way or another, I thank . . . Tom
and John for shooting the bull; Frank, Vlada, Sharon, and Ronda for holding things
together the first years; Eric for teaching me that nothing lethal grows in home brew;
Darrin, Rom, and Phil for alternate realities; Jim, Ross, Wiz, Elise, Scott, Bob, Abby,
and Lynda for reminding me that old friendships are worth maintaining; a family that

was always there; and Antje, without whom the rest matters little.

List of Tables
List of Figures
List of Abbreviations

General Introduction

TABLE OF CONTENTS
Page
. . .viii
. . .ix

...xii

Chapter 1: Cloning of a cDNA encoding l—aminocyclo-
propane-l-carboxylate oxidase from pea

Abstract

Introduction

Materials and Methods

Results

Discussion

References

..12

..18

...26

Chapter 2: Localization of l-aminocyclopropane-l—
carboxylate oxidase from tomato in

transgenic yeast
Abstract
Introduction
Materials and Methods
Results
Discussion

References

...31

...32

...33

...36

...45

...48

vi

Chapter 3: Sequential induction of the ethylene
biosynthetic enzymes by indole-3—
acetic acid in etiolated peas

Abstract . . .52
Introduction . . .53
Materials and Methods ...56
Results . . .63
Discussion . . .77
References . . .82

Chapter 4: Cloning of two cDNAs encoding l-aminocyclo-
propane-l-carboxylate synthase that are
induced by indole-3-acetic in etiolated

peas

Abstract . . .86
Introduction . . .87
Materials and Methods ...89
Results . . .92
Discussion ... 1 14
References .. . 124

Conclusions .. . 127

vii

LIST OF TABLES.

Page
Table 1.1. ACC oxidase activity in transgenic yeast. ...15
Table 1.2. Effect of inhibitors on ACC oxidase activity in transgenic
yeast. .. .16

Table 2.1. Ethylene-forming activities of cells, protoplasts, and
vacuoles. ' . . .41

viii

Figure 1.1.

Figure 1.2.
Figure 1.3.

Figure 1.4.

Figure 1.5.

Figure 1.6.

Figure 2.1.

Figure 2.2.

Figure 2.3.

Figure 3.1.

Figure 3.2.

Figure 3.3.

Figure 3.4.

LIST OF FIGURES

Nucleotide and deduced amino acid sequence of a pea ACC
oxidase cDNA clone PE8.

Immunoblot analysis of protein extracts from transgenic yeast.
Amino acid sequence alignment of ACC oxidases.

Amino acid alignment of consensus sequences of ACC oxidases
and dioxygenases from plants.

Amino acid consensus sequences from the carboxy-terminal
portions of plant dioxygenases.

Amino acid alignment of sequence surrounding the lysine
residue which binds C02 in Rubisco with consensus sequence
of ACC oxidase.

Time course of induction of ACC oxidase in transgenic yeast
(TF-808-l) following transfer to galactose medium.

Immunoblot analysis of protein extracts from yeast cells,
protoplasts, and vacuoles.

Immunoblot analysis of proteins from differential centrifugation
and Endo-H treatment.

Nucleotide and deduced amino acid sequence of ACC synthase
cDNA clone PS-ACS2.

Effect of ethylene on ACC oxidase transcript abundance and
enzyme activity.

Effect of IAA on ACC synthase activity and rate of ethylene
production in the first intemode of etiolated pea seedlings.

Effect of IAA and NBD on ACC oxidase transcript abundance
and enzyme activity.

ix

Page

..13
..17

...20

...22

...24

...25

...39

...42

...44

...62

...66

...68

Figure 3.5.

Figure 3.6.

Figure 3.7.

Figure 3.8.

Figure 3.9.

Figure 4.1.
Figure 4.2.

Figure 4.3.

Figure 4.4.
Figure 4.5.
Figure 4.6.
Figure 4.7.
Figure 4.8.
Figure 4.9.
Figure 4.10.
Figure 4.11.
Figure 4.12.

Figure 4.13.

Figure 4.14.

Competitive effects of NBD and ethylene on ACC oxidase
transcript abundance and enzyme activity during IAA treatment.

Comparison of rates of accumulation of ACC synthase and ACC
oxidase transcript and enzyme activity after auxin treatment.

Effect of ethylene pretreatment on rate of auxin-induced
ethylene production.

Effect of ethylene pretreatment on auxin-induced ACC synthase
activity and transcript abundance.

Positive feedback of ethylene on ACC oxidase after auxin
treatment.

Nucleotide and deduced amino acid sequence of PS-ACSl.
3’ ends of eight PS-ACSl clones.

Predicted hairpin structure formed by base pairs 978 to
1035 of PS-ACSl.

Nucleotide and deduced amino acid sequence of PS-ACS2.
Cross-hybridization of PS-ACSl and PS-ACSZ.

Effect of auxin and ethylene on ACC synthase activity.

Effect of auxin and ethylene on ACC synthase transcript levels.
Expression pattern of two transcripts hybridizing to PS-ACSl.
Genomic DNA blot probed with PS-ACSl.

RNA blot analysis using 3’-UTR of PS-ACSl.

Diagram of the procedure for RNase H mapping.

RNA blot analysis after removal of poly(A)-tail.

Oligonucleotide-directed RNase H mapping of the PS-ACSl
transcript.

Amino acid sequence comparison of ACC synthase clones similar
to LE-ACS3.
x

...70

...73

...75

...76

...80

...94

...96

...97

...98

...99

...101

...102

...105

...106

...107

...109

..110

...113

...117

Figure 4.15. Amino acid sequence comparison of ACC synthase clones similar
to LE-ACSZ. 120

Figure 5.1. Positive and negative fwdback of ethylene on the biosynthetic
pathway after auxin treatment. ...128

xi

LIST OF ABBREVIATIONS

ACC l-aminocyclopropane-l-carboxylate
IAA indole-3-acetic acid

IPNS isopenicillin N synthase

NBD 2,5-norbomadiene

PCR polymerase chain reaction

Rubisco ribulose-l ,5-bisphosphate carboxylase/oxygenase
SAM S-adenosyl-L—methionine

UTR untranslated region

GENERAL INTRODUCTION

Ethylene controls a wide range of physiological and developmental processes
including ripening of fruit, abscission, senescence, and responses to wounding and auxin
(Abeles et al., 1992). Most of these ethylene effects are associated with changes in
ethylene production. Thus, defining the regulation of ethylene biosynthesis is of great
importance in understanding the role of ethylene in growth and development.

In higher plants, ethylene is produced from methionine via S-adenosyl-L-
methionine (AdoMet) and the cyclic, nonprotein amino acid l-aminocyclopropane-l-
carboxylic acid (ACC). The formation of AdoMet from methionine is catalyzed by
AdoMet synthase. Besides being an intermediate of ethylene biosynthesis, AdoMet acts
as a methyl donor in a number of biosynthetic reactions and as an intermediate in
polyamine bioysynthesis. The first committed step of ethylene biosynthesis, the
formation of ACC from AdoMet, is catalyzed by ACC synthase (Abeles et al., 1992).
The 5’-methy1thioadenosine released during this reaction enters a modified methionine
cycle which maintains methionine levels even during periods of high ethylene production
(Miyazaki and Yang, 1987). The final enzyme, ACC oxidase, converts ACC to ethylene.
Besides being converted to ethylene, ACC is metabolized to 1-(malony1amino)cyclo—
propane-l-carboxylic acid (MACC), permanently removing the ACC from the ethylene
biosynthetic pathway under most physiological conditions (Yang and Hoffman, 1984).

Ethylene regulates its own biosynthesis. In ripening fruits and senscing tissues,

2

ethylene production is autocatalytic. In vegetative tissue, however, ethylene both
positively and negatively regulates its own biosynthesis (Abeles et al., 1992). Ethylene
stimulates the accumulation of ACC oxidase activity in citrus leaves (Riov and Yang,
1982), tobacco leaves (Chalutz et al., 1984), and in etiolated pea stems (Schierle et al.,
1989). Autoinhibition of ethylene seems to be directed primarily at ACC synthase.
Ethylene partially suppresses the increase in extractable ACC synthase activity in indole-
3-acetic acid (IAA)-treated mung bean hypocotyl sections (Yoshii and Imaseki, 1982).
In etiolated pea stems, propylene (an ethylene analog) completely inhibits wound—induced
ethylene accumulation (Saltveit and Dilley, 1979).

The goal of this dissertation was to examine the role of negative and positive
feedback on IAA-induced ethylene production in etiolated pea stems. It is now known
that multiple ACC synthase genes responsive to either IAA or wounding would have been
induced under the conditions used in the previous IAA-induction experiments. Gene
specific probes can determine if expression of some or all of the genes expressed after
auxin treatment are negatively regulated by ethylene. Molecular probes also allow
greater sensitivity in detecting rapid and subtle changes in ACC synthase and ACC
oxidase transcript levels after IAA treatment. This sensitivity is especially important in
examining the role of ethylene-induced ACC oxidase activity in the regulation of ethylene

production.

References

Abeles F, Morgan PW, Saltveit ME (1992) Ethylene in Plant Biology. Academic Press,
New York

Chalutz E, Mattoo AK, Solomos T, Anderson JD (1984) Enhancement by ethylene of
cellulysin-induced ethylene production by tobacco leaf discs. Plant Physiol 74: 99-
103

Miyazaki JH, Yang SF (1987) The methionine salvage pathway in relation to ethylene
and polyamine biosynthesis. Plant Physiol 69: 366-370

Riov J, Yang SF (1982) Effects of exogenous ethylene on ethylene production in citrus
leaf tissue. Plant Physiol 70: 136—141

Schierle J, Rohwer F, Bopp M (1989) Distribution of ethylene synthesis along the
etiolated pea shoot and its regulation by ethylene. J Plant Physiol 134: 331-337

Yang SF, Hoffmann NA (1984) Ethylene biosynthesis and its regulation in higher
plants. Annu Rev Plant Physiol 35: 155-189

Yoshii H, Imaseki H (1982) Regulation of auxin-induced ethylene biosynthesis.
Repression of inductive formation of 1-aminocyclopropane-l—carboxylate synthase
by ethylene. Plant & Cell Physiol 23: 639-649

CHAPTER 1

CLONING OF A cDNA ENCODING
l-AMINOCYCLOPROPANE-l-CARBOXYLATE OXIDASE FROM PEA

Abstract

A clone for 1-aminocyclopropane—l—carboxylate (ACC) oxidase was isolated from a
cDNA library made from poly(A)+ RNA of apical hooks of etiolated peas treated for 4
h with 10“ M IAA and was sequenced. The 1122-bp insert was subcloned into a yeast
expression vector which was then used to transform yeast strain F808. When induced
with galactose, transgenic yeast with the insert in the coding orientation was able to
convert ACC to ethylene, and the conversion was inhibited by CoCl2 and aminoiso-
butyrate, two inhibitors of ACC oxidase activity. By immunoblot analysis, it was shown
that polyclonal antibodies raised against a tomato ACC oxidase recognized a protein of
the expected size in transgenic yeast if the insert was in the coding orientation but not
if it was in a non-coding orientation. These results confirm that the cDNA clone encodes

a functional ACC oxidase from peas.

Introduction

The final step of ethylene biosynthesis, the conversion of ACC to ethylene, is
catalyzed by ACC oxidase (also referred to as the "ethylene-forming enzyme, " EFE).
Knowledge about ACC oxidase remained limited long after the pathway of ethylene
biosynthesis had been elucidated (see Kende, 1993). Attempts to purify and characterize
the enzyme failed because enzymatic activity disappeared when the tissue was
homogenized. While a number of cell-free systems were capable of converting ACC to
ethylene, they all lacked the stereospecificity exhibited by the in viva ethylene-forming
system with respect to the conversion of 2-ethyl-ACC stereoisomers to l-butene (Yang
and Hoffman, 1984).

The enzyme was finally identified by the characterization of a ripening-related
cDNA clone from tomato fruits. Slater et al. (1985) isolated a number of cDNA clones
corresponding to mRNAs that were present in ripe but not in mature green tomato fruits.
One of these clones, pTOM13, coded for a 35-kDa protein whose expression is
correlated with increased ethylene synthesis (Smith et al. , 1986). Tomato plants
transformed with an antisense construct of pTOM13 had greatly reduced ethylene
biosynthesis in wounded leaves and ripening fruits (Hamilton et al. , 1990). The level
of reduction in ACC oxidase activity was related to the number of copies of antisense
pTOM13 genes (Hamilton et al., 1990). Based on these results, Hamilton et al. (1990)
suggested that pTOM13 encoded ACC oxidase. Final proof came when a corrected

version of pTOM13 containing the full 5’ end was expressed in yeast and was shown to

7
possess stereospecific ACC oxidase activity (Hamilton et al., 1991). Independent

confirmation came from the injection of mRNA for pHTOMS, another cDNA from
tomato with 88% nucleotide identity with pTOMl3, into oocytes of Xenopus laevis.
Expression of pHTOMS conferred ACC oxidase activity that also met the stereospecifici-
ty requirements (Spanu et al., 1991).

The identification of the cDNA for ACC oxidase in tomato led to the isolation of
a number of cDNA clones for ACC oxidase from other species (see Zarembinski and
Theologis, 1994). These new cDN A clones were identified solely by their sequence
similarity to the two tomato clones. While these clones did have a high degree of
identity to pTOMl3, they also shared significant identity with other dioxygenase-related
cDNAs, such as E8 (Deikman et al., 1988).

Because the deduced amino acid sequence of pTOM13 showed 58% similarity to
the amino acid sequence of flavanone 3-hydroxylase, a 2-oxoglutarate—dependent
dioxygenase (Hamilton et al., 1990), Ververidis and John (1991) extracted and assayed
ACC oxidase activity from melon fruits using conditions that had been shown to preserve
flavanone 3-hydroxylase activity from Petunia hybrida. By extracting the enzyme under
N2 gas and including Fe2+ and ascorbate in the assay medium, they completely
reconstituted ACC oxidase activity that displayed the proper stereospecificity in vitro.
Previous failures to isolate ACC oxidase activity can be attributed to the dilution of the
essential cofactors Fe2+ and ascorbate during homogenization. Despite the sequence
similarity to oxoglutarate-dependent dioxygenases, ACC oxidase does not require 2-

oxoglutarate as a cofactor (Smith et al., 1992).

A unique feature of ACC oxidase is its requirement of CO2 for enzyme activity
(Dong et al., 1992). In a number of plant systems, ACC oxidase activity is enhanced
by high levels of C02 (Yang and Hoffman, 1984). Working with purified enzyme, Dong
et al. ( 1992) found that in vitro activity is completely abolished if C02 is trapped with
KOH. They also determined that half-maximum activity is reached at 0.5% CO; and
maximum activity at 2-4% C02. By analogy to the activation of ribulose-1,5-bis-
phosphate carboxylase/oxygenase (Rubisco; Iorimer and Miziorko, 1980) and urease
(Park and Hausinger, 1995), the CO2 is predicted to form a carbamate with the e-amino
group of a lysine residue. This carbamate would interact with a cation, Fe“ in the case
of ACC oxidase, to bind the metal and facilitate its incorporation into the catalytic site.
Although an aspartate or glutamate residue should, theoretically, be able to perform a
similar role, crystal structure data of urease (Jabri et al., 1995) suggests that the added
length of a carbamate on a lysine residue is necessary to span the distance to the active
site.

Work with ACC oxidase reported below has been separated into two parts: (i)
cloning of a cDNA encoding an ACC oxidase that is expressed in the stem of etiolated
pea seedlings; and (ii) confirming that the insert of the clone encoded a functional ACC

oxidase by expression of the cDNA in transgenic yeast.

Materials and Methods

Cloning and sequencing of ACC oxidase cDNA

From a comparison of nucleotide sequences of pTOM13 from tomato (Holdsworth
et al. , 1987) and an ACC oxidase-related cDNA from avocado (McGarvey et al. , 1990),
two conserved regions of FGTKVSN and PKEPRFE were selected for the synthesis of
non-degenerate oligonucleotideprimers HK-21 , 5 ’ -TITGGTACTAAAGTTAGCAAC-3 ’ ,
and HK-20, 5’—G(GAATI‘C)AAATC'ITGGCTCTITGGC-3’ (nucleotides in parenthesis
encode an EcoRI site), corresponding to the respective pTOM13 sequences. These
primers were used for a polymerase chain reaction (PCR)-based amplification from a
Lambda Zap 11 cDNA library (Stratagene, La Jolla, CA, USA) made from poly(A)+
RNA from apical hooks of etiolated peas treated for 4 h with 104 M IAA. Thermo-
cycling was performed at 94°C for 1 min, 37°C for 1 min, and 72°C for 1 min, for 30
cycles. The PCR product (PEl) was sequenced to confirm its similarity to pTOM13 and
then used to screen the pea cDNA library. Hybridization was done in 6X SSC, 0.1%
SDS, and 5X Denhardt’s Reagent at 62°C. The final wash was performed at 0.5X SSC,
0.1% SDS at 62°C. Both strands of the insert of the longest full-length cDNA, pPE8,

were sequenced.

Production of transgenic yeast
The full-length cDNA insert was excised from pBluescript SK‘ (Stratagene, La

J olla, CA) by EcoRI digestion, recovered by electrophoresis onto DE—81 paper

10

(Sambrook et al., 1989), and ligated into a yeast expression vector with a galactose-
inducible promoter, pYE82.0 (Invitrogen, San Diego, CA), to yield pYPE8. Yeast strain
F808 (MA Ta/oz GAL] + leu2-3 leu2-112 his4-519 adel -100 ura3-52) was transformed by
electroporation (Bio-Rad Technical Bulletin) with pYPE8 to yield TYPE8—F808.

Transformants were selected by their ability to grow on a medium that lacked uracil.

Culture conditions for transgenic yeast

Yeast was grown on a shaker at 30°C to mid-log phase in a medium containing
0.65% yeast nitrogen base without ammonium sulfate or amino acids (DIFCO, Detroit,
MI), 2% galactose, 0.5% ammonium sulfate, 0.002% adenine sulfate, and 470 mg/L of
an amino acid mixture consisting of 20 mg arginine, 100 mg aspartate, 60 mg histidine,
60 mg leucine, 30 mg lysine, 60 mg methionine, 50 mg phenylalanine, 60 mg threonine,

40 mg tryptophan, and 30 mg tyrosine.

Measuring ACC oxidase activity in transgenic yeast

To measure ACC oxidase activity, 690 pL of mid-log cells, 100 uL l M Tris-
HCl, pH 7.2, 100 ILL 100 mM ACC (Sigma Chemical Co., St. Louis, MO), 100 ILL 300
mM Na-ascorbate, and 10 uL 0.1 mM FeSO4 were mixed in 4—mL vials which were
capped and incubated on a shaker at 30°C for 1 h. Ethylene concentrations were
determined by analyzing 1 mL of the headspace in a gas chromatograph (GC). The
number of cells per reaction was determined by counting on a hemocytometer. In the

inhibitor studies, the inhibitors were resuspended in the above Tris buffer and added to

11

the mixture such that the other volumes were not affected.

Gel electrophoresis and immunoblotting

To isolate protein, yeast cells were collected by centrifugation at 600 x g for 15
min in microcentrifuge tubes. The pelleted cells were washed in ice-cold extraction
buffer [50 mM Tris—2-N-morpholinoethanesulfonic acid (MES), pH 7.8, 2 mM
dithiothreitol (DT'I‘), 1 mM PMSF, 250 mM sucrose]. The pellet was then suspended
in an equal volume of ice-cold extraction buffer and an equal volume of glass beads
(diameter: 0.45 mm). The tubes were shaken vigorously on a Vortex mixer for 10 min
at 4°C. The tubes were punctured at the bottom, placed inside fresh tubes, and
centrifuged at 100 x g to separate the extract from the glass beads. The supernatant from
the lower tube was transferred to a fresh tube and centrifuged again at 100 x g for 15
min at 4°C to remove remaining cellular debris. The supernatant was combined with
0.25 volume of 4X sample buffer (1X sample buffer is 0.03 M Tris—HCl, pH 6.8, 1%
SDS, 5% glycerol, 0.0125% bromophenol blue) and boiled for 10 min.

Samples were subjected to electrophoresis at 50 V in 10% SDS-polyacrylamide
gels (Laemmli, 1970) with l-mm spacers using the Bio-Rad Mini-Protean II apparatus
(Bio-Rad Laboratories, Richmond, CA). Transfer to 0.45 am nitrocellulose (Schleicher
and Schuell, Keene, NH) was performed at 40 V for 3 h. For immunoblot detection of
the protein, a secondary antibody conjugated to alkaline phosphatase was used as

described by Bumette (1981).

12
Results

Cloning of ACC oxidase

To isolate a cDNA clone for ACC oxidase, a PCR product was obtained from a
pea cDNA library made from poly(A)+RNA isolated from etiolated pea hooks treated for
4 h with 10" M IAA using non-degenerate oligonucleotide primers based on the
nucleotide sequence of a tomato ACC oxidase, pTOM13. The PCR product, PEl, was
used to screen the same cDNA library for a full-length clone. From the 300,000 plaques
screened, 26 independent clones were isolated. Of these, eight clones were sequenced
and found to be identical except for different lengths at the 5’ and 3’ ends. The longest
clone, pPE8, contained an insert of 1122 bp encoding an open reading frame of 317
amino acids with a predicted mass of 36 kDa (Figure 1.1). The deduced amino acid
sequence shared 71% and 69% identity with those of two tomato ACC oxidase cDNAs

(Hamilton et al., 1991; Spanu et al., 1991, respectively).

13

1 AAAATGGAAAACTTTCCAATTGTTGACATGGGGAAGCTTAACACAGAAGACAGAAAATCA
M E N F P I V D M G K L N T E D R K S
61 ACCATGGAGTTGATCAAAGATGCCTGTGAGAACTGGGGTTTCTTTGAGTGTGTGAATCAT
T M E L I K D A C E N W G F F E C V N H
121 GGTATATCTATTGAGATGATGGATACGGTTGAGAAGCTGACAAAGGAACACTACAAGAAG
G I S I E M M D T V E K L T K E H Y K K
181 TGTATGGAACAAAGGTTCAAAGAAATGGTTGCAACCAAAGGTTTGGAGTGTGTTCAGTCA
C M E Q R F K E M V A T K G L E C V Q S
241 GAGATAGATGATTTGGATTGGGAAAGCACTTTCTTTTTGCGCCATCTTCCTGTTTCCAGT
E I D D L D W E S T F F L R H L P V S S
301 ATCTCAGAGATCCCAGATCTTGATGATGATTACAGGAAGGTGATGAAGGAATTTGCGTTG
I S E I P D L D D D Y R K V M K E F A L
361 AAATTGGAAGAACTTGCGGAGGAACTTCTTGACTTGTTGTGTGAGAATCTTGGGCTTGAG
X L E E L A E E L L D L L C E N L G L E
421 AAAGGGTATTTGAAGAAGGCTTTTTATGGATCAAAGGGTCCAAATTTIGGGACGAAAGTT
K G Y L K K A F Y G S K G P N F G T K V
481 AGTAACTACCCTCCTTGTCCTAAACCGGAACTCATCAAGGGACTTAGAGCTCACACAGAT
S N Y P P C P K P E L I K G L R A H T D
541 GCCGGCGGAATCATTCTTCTCTTCCAAGATGACAAAGTCAGTGGACTTCAGCTTCTCAAA
A G G I I L L E Q D D K V S G L Q L L K
601 GATGACCAATGGATTGATGTCCCTCCAATGCGTCACTCTATTGTCATCAATCTTGGTGAT
D D Q W I D V P P M R H S I V I N L G D
661 CAACTCGAGGTGATAACCAATGGAAAGTACAAGAGTGTGATGCATAGAGTAATAGCACAA
Q L E V I T N G K Y K S V M H R V I A Q
721 ACAGATGGTGCTAGAATGTCCATAGCATCCTTCTACAATCCAGGGGATGATGCTGTGATT
T D G A R M S I A S F Y N P G D D A V I
781 TCACCAGCTTCAACCTTATTGAAGGAAAATGAAACAAGTGAAGTTTATCCAAAGTTTGTG
S P A S T L L K E N E T S E V Y P K F V
841 TTTGATGATTACATGAAACTCTACATGGGACTCAAGTTCCAAGCTAAAGAGCCTAGATTT
F D D Y M K L Y M G L K F Q A K E P R F
901 QAAGCTATGATGAAAGCCATGTCAAGTGTTAAAGTGGGACCAGTAGTAAGCATATAGTTA
E A M M K A M S S V K V G P V V S I *
961 TGTGTGTGGTTTATATTACTAAGGTATTATTAATGTTTGGTTGGTGTTTTAAGATTGTGC
1021 AGTGTCTAAAGTGTTATATTAAAAAAAATAAAGTATGATCTTAGATTGTGTGTGACAATT
1081 AATCAGTTATGATTAAGTGATTAAGTTTATGTAATGCGATGC

Figure 1.1. Nucleotide and deduced amino acid sequence of a pea ACC
oxidase cDNA clone PE8 (Peck et al., 1993). The primer sites used to amplify
PE1 are underlined.

14

Expression of pPEB in transgenic yeast

To determine whether the pPE8 insert encoded a functional ACC oxidase,
the insert was cloned into yeast expression vector pYESZ.0 (Invitrogen, San
Diego, CA, USA) and transformed into yeast strain F808 to create TYPE8-
F808. Expression of the insert was controlled by a galactose-inducible
promoter. When grown under inductive conditions, transgenic cells contained
ACC oxidase activity as determined by their ability to convert ACC to ethylene
(Table 1.1). Yeast cells containing a pYE82.0 plasmid with the PE8 insert in
the opposite orientation (TY8EP-F808) did not produce ethylene above
background level (medium only; Table 1.1). ACC oxidase activity in the
transgenic cells was reduced by CoClz, a potent inhibitor of ACC oxidase
activity, and aminoisobuterate (AIB), a weak inhibitor (Table 1.2). A transgenic
protein with the expected mass of 36 kDa was recognized by immunoblot
detection (Figure 1.2) using antibodies raised to tomato ACC oxidase (Peck et
al., 1992). TYPE8—F808 cells grown under non-inductive conditions did not
produce ethylene from ACC above background levels and did not accumulate
antigenic protein (data not shown). These results confirm that the clone PE8

encodes a functional ACC oxidase.

15

Table 1.1: ACC oxidase activity in transgenic yeast. Yeast containing
plasmids with inserts in the coding orientation (TYPE8-F808) and in the non-
coding orientation (TY8EP—F808) were grown under inductive conditions.

Values are the average of duplicate samples. Experiments were performed
twice with similar results.

 

Ethylene Production
(pmol h“1 10" cells)

TYPE8-F808 1371.2
TY8EP-F808 19.1

Medium Only 12.8

16

Table 1.2: Effect of inhibitors on ACC oxidase activity in transgenic yeast.
Values are averages of duplicate samples. Experiments were performed twice
with similar results.

 

 

ACC COCIZ AIB Ethylene Production
(mM) (mM) (mM) (pmol h'1 10'6 cells)
10 - - 1658.3

10 1 - 118.8

10 5 - 12.2

10 10 - 5.4

1 - - 515.7

1 - 10 365.9

1 - 50 279.5

1 - 100 229.1

 

l7

‘—--' ...—r
##4-

 

Figure 1.2. Immunoblot analysis of protein extracts from transgenic yeast.
Yeast containing the cDNA insert PE8 in the non-coding orientation (lane 1) and
in the coding orientation (lane 2) were induced by galactose for 24 h. Bio-Rad
molecular weight markers are in lane M. The arrow marks the expected size

of ACC oxidase.

18
Discussion

Analysis of deduced amino acid sequences is often useful in providing
insights into the structure and reaction mechanism of enzymes. A number of
reviewers have presented sequence alignments between ACC oxidases and
related dioxygenases (Prescott, 1993; Zarembinski and Theologis, 1994).
These alignments were structured to highlight identical residues. While this
type of analysis is helpful in determining common dOmains, such as binding
sites for Fe“ and ascorbate, it will not identify domains unique to ACC
oxidase. The analysis presented below was designed to supplement these
previous alignments by focusing on regions conserved in ACC oxidases but not
in other dioxygenases.

As seen in Figure 1.3, all known ACC oxidases share 46% identity and
64% similarity at the amino acid level. The consensus sequence derived from
this comparison facilitates comparison with other dioxygenases. As pointed
out by Prescott (1993), dioxygenases from animals, plants, and microorganisms
share suprisingly little similarity. It may be more meaningful, therefore, to
compare the consensus sequence of ACC oxidase with a consensus sequence

Obtained only from plant dioxygenases (Figure 1.4).

19

Legend for Figure 1.3. Sequences are from pea (Peck et al., 1993), tomato
(Kock et al., 1991; Spanu et al., 1991), avocado (McGarvey et al., 1990),
carnation (Wang and Woodson, 1991), apple (Dong et al., 1992b), petunia
(Tang et al., 1993), peach (Callahan et al., 1992), Arabidopsis (Gomez-Lim et
al., 1993), and orchid (Nadeau et al., 1993). Identical residues aligned below
the pea sequence are shown by an asterisks (*l. Gaps in the alignment are
shown by periods (.).

PEA
TOMATOI
TOMATOZ
AVOCADO
CARNATION
APPLE
PETUNIA
PEACH
ARAB.
ORCHID

PEA
TOMATO
TOMAT02
AVOCADO
CARNATION
APPLE
PETUNIA
PEACH
ARAB.
ORCHI

PEA
TOMATO
TOMATOZ
AVOCADO
CARNATION
APPLE
PETUNIA
PEACH
ARAB.
ORCHID

PEA
TOMATO
TOMATOZ
AVOCADO
CARNATION
APPLE
PETUNIA
PEACH
ARAB.
ORCHID

PEA
TOMATO
TOMAT02
AVOCADO
CARNATION
APPLE
PETUNIA
PEACH
ARAB.
ORCHID

Figure 1.3. Amino acid sequence alignment of ACC oxidases.

MEN...FPIV

eta...***1
***...***I
*DS...**VI
*A*IVN***I
*AT...**VV
new...***1
***...G***
*es...****

**S.GS**VI

MMDTVEKLTK
V******M**

V**R******
L**E**R***
L**K**RM**
LL*****M**
V******M**
FL****R***
LL*K***M**
L*NR**TVE*

LPVSSISEIP
**T*N**QV*
**T*N**QV*
****NL****
R*T*N***V*
**S*N*****
**I*N***V*
**K*N***V*
**V*N**DV*
**T*N**Q**

KAFYGSK.G.
“******.*.

u******.*.
M**A*TTT*L
N****AN.*.
*v*****.*.
u******.*.
*****TN.*.
*V*****.R.

*V*C*GSD*L

SGLQLLKDDQ

********E*
********E*
A*******GE
********GH
********GE
********G*
*eae****gw
********GE
********GE

DMGKLN...T
NLE***...G
NLEN**...G
N*E**E...G
**E***NYNG
*LSLV*...G
SLD*V*...G
N*EG**...G
NLE*D*...G
N*EL*Q...G

EHYKKCMEQR
c*********

G*********
**********
*****FR**K
D****T****
g*********
***RQ*L***
********E*

***RRFR***

DLDDDYRKVM
***EE**E**

***EE**E**
**T*EH****
****Q***L*
**EEE***T*
***EE**E**
**E*Q**N**
*******TL*
*****C*ST*

PNFGTKVSNY
**********

**********
*T********
*T********
**********
**********
*T********
*T********
*T********

WIDVPPMRHS

**********
**********
*v*****u**
*v*****x**
*V*****H**
**********
Q*L*******
*V****VK**
******v***

20

EDRKSTMELI
DE*AN***M*
DE*AK***M*
QE*AA**K**
VE*SLVLDQ*
*E*AA*L*K*
vgeAAtewna
*G**A***K*
*E*AI***K*
SQ*PAA*A*L

FKEMVATKGL
***L**S***

***L**S***
***LM*S*.V
**D**Q****
******A***
***L**S*A*
***L**S***
***SIKNR**
***FASKTLD

KEFALKLEEL
RD**KR**K*
RD**KR**K*
****E***K*
****AQI*R*
****VE**K*
RD**KR**K*
**********
*D**G*I*K*
*S***E**N*

PPCPKPELIK
******D***

******D***
****R*E*F*
******D***
******D***
******D***
**********
****N*D*V*
******D**N

IVINLGDQLE

**v*******
**v*******
********v*
**v*******
********I*
**v*******
**********
**v*******
**v*1*****

KDACENWGFF

**********
**********
u*********
****H*****
u*********
**********
**********
**********
R*******LY

ECVQSEIDDL
*A**A*VT**
*A**A*VT**

'*GAVVDAN*M

VSAE*QVN*I
DD*****H**
*G**A*VT*M
*A*KT*VN*M
DSLR*SVN*V
TVENV*PEN*

AEELLDLLCE
**********

**********
**QV******
5*Q*******
**K*******
**********
**********
s*********
**R*******

GLRAHTDAGG
**********

**********
**********
**********
*****s****
******we**
**********
**********
*****s****

VITNGKYKSV

**********
**********
**********
******t***
**********
**********
**********
**********
**********

ECVNHGISIE
*L*****pH*

*L*****PH*
*L***S*PV*
QV***SL*H*
*L****M*T*
*L*****PR*
*L*S***PT*
********L*
*LL*****H*

DWESTFFLRH
**********

**********
*******I**
******y***
**********
********K*
******y***
******y*K*
**********

NLGLEKGYLK
**********

**********
**********
******A***
**********
**********
**********
**********
D*********

IILLFQDDKV
**********

**********
L*******R*
**********
**********
**********
**********
**********
**********

MHRVIAQTDG

L*********
**********
****v*****
**********
*******s**
*******K**
E*********
E***LS****
L***V*****

Figure 1.3 (continued)

PEA
TOMATO
TOMAT02
AVOCADO
CARNATION
APPLE
PETUNIA
PEACH
ARAB.
ORCHID

PEA
TOMATO
TOMAT02
AVOCADO
CARNATION
APPLE
PETUNIA
PEACH
ARAB.
ORCHI

.ARMSIASFY
.T***L****

.T***L****
.N***L****
.N********
.T********
.****L****
.T********
EG********

.N****SA**

LYMGLKFQAK

**3*******
*ea*****p*
**A*******
**LK****ED
**s*******
**A*******
**M*****p*
**SAV*****

**IRK**E*D

NPGDDAVISP
*aeseetwy*

*ten****y*
***S****F*
***3****y*
***N*SF***
***s****y*
***s****y*
***s*s**p*
***S****F*

EPRFEAM.MK
*******..*
*******..*
*****V*KM*
*******..
*******..
*******..
*******..
*******..

*******.

*I-Irfiifi

21

ASTLL.KENE
*K**VE**A*
*PS*I....*
*PA*VE**A*
*P**VE**.*
*PAV*....*
*PA*VE**A*

*P**VE**A*'

VPE*IG**A*
*pA*VE**A*

AMSSV..KVG
**E*D.....
**EANVEL*D
*VETA..NLS
**ETT....*
*KE*T.....
**ETD.V*MD
*VETN.ISL*
**ETTVANNV
S*EI*MSSQ.

T.....SEVY
ES....TQ**
ES....KQ**
EK....K***
EK....CRA*
KKTED.APT*
EN....KQ**
EK....NQ**
.K.EK.K*N*
EKEEKKK*I*

..PVVSI
..*IA*A
..QIA*A
..*ITT*
..**PTA
..**ATA
..*IATV
..*IATA
G.*LATA
..*IPTA

PKFVFDDYMK

**********
**********
*****E***N
*****E***N
**********
**********
*****E****
*R***E****
*****Q***N

CONSENSUS
DIOXY.

CONSENSUS
DIOXY.

CONSENSUS
DIOXY.

CONSENSUS
DIOXY.

CONSENSUS
DIOXY.

CONSENSUS
DIOXY.

CONSENSUS
DIOXY.

Figure 1 .4.

1994; Xu et al.,
Meldgaard,

1.3 ------ P11

1

51
51

101
101

151
151

201
201

251
251

301

M--//-vP-

vm--VE-m-K
1i --------

-P-S-iS-iP
_-_-//-__-

--F-G-----

--f-_-_//_

.......
......

.......
......

22

-HYkk--Eqr
-FF-1P---K

DL-ee-R--M
-------- v—

P—FGTKVSNg
----v-1Nf§

wi-VPgm-Hs
w-sv-g---A

NfiG-DavI-g

 

FKe -
—rv ———————

k-FA--lE-L
--y---l--1

......
.........

.........
..........
.........

- 1
"// ......
aE-ILDLLCE
e--im-11--

......

 

......
'''''''''''''

vrrgcxxxsv

e-vNfigi--E
-vv-Hgv--e

DgEsrrfer
_g_e_———-—

-LeLangLx
ale-//y-k

.........

-§Rv1aQ-DG
-§rav -----

PkFVFdDYMk

 

Amino acid alignment of consensus sequences of ACC oxidases
(from Figure 1.3) and dioxygenases from plants: GA-20 oxidases (Lang et al.,

1992).

1995).

flavanone 3-hydroxylases (Britsch et al.,
hyoscyamine 6-hydroxylase (Matsuda et al.,
Identical residues are in upper case.

1992:
1991).

Functionally conserved residues are in

lower case. Shaded residues are absolutely conserved in all ACC oxidases and
dioxygenases from this comparison. Double backslashes (//) indicate regions
of variable length.

23

Similarity with other dioxygenases ends at Pr0270. In fact, similarity between
all dioxygenases ends at that same proline. However, at various distances after
that residue, enzymes compared by substrate specificity have extremely well-
conserved domains (Figure 1.5). The high degree of identity suggests that
these residues may be involved in the binding of the respective substrates of
these enzymes. The crystal structure data of isopenicillin N synthase (IPNS)
indicate that the C-terminal tail will fold to enter the active site region,
supporting this hypothesis.

Another unique feature of ACC oxidase activity is its absolute require-
ment for CO2 (Dong et al., 1992a). By analogy to Rubisco (Lorimer and
Miziorko, 1980) and urease (Park and Hausinger, 1995), the only other known
COz-activated enzymes, it is believed that CO, forms a carbamate with the 6-
amino group of a lysine residue. This carbamate binds the cation and brings it
into the catalytic site. Sequence comparison between ACC oxidase and
Rubisco suggests a candidate lysine for this activation step. LysZOZ is the
residue involved in the formation of the carbamate in Rubisco (Lorimer, 1981).
The consensus sequence around this residue agrees well with the consensus
sequence around Ly3208 from ACC oxidase (Figure 1.6). Future experiments
will be directed at determining which amino acid residue interacts with 602 to

activate ACC oxidase.

24

ACC Oxidases: 29°YPkFVFdDYMkLY—--KFq--EPRFE-M--Ka
CIA-20 Oxidases: 34st-W-mlLEf-QKhYFI-D-nTL-aF--Wl
Flavanone-3-hyd roxylase: 319eEF’ITFaEMYFIRKM-I'DLeLAr-KKQAKeQ-MQ

Figure 1.5. Amino acid consensus sequences from the carboxy-terminal
portions of plant dioxygenases. Alignments were derived from sequences cited
in Figure 1.4. Identical residues are shown in upper case while functionally
conserved residues are shown in lower case. Numbering of residues is based
on consensus alignments.

25

ACC Oxidase: sGLQI-L ”Kit-“”1”???“
RuBISCO: can)” ”3993mm”

Figure 1.6. Amino acid alignment of sequence surrounding the lysine residue
which binds 002 in Rubisco (Lorimer, 1981) with consensus sequence of ACC
oxidase. Identical residues are shown in upper case while functionally
conserved residues are shown in lower case. Shaded residues are identical in
both enzymes. Residues that are similar are connected by two periods (z).

i

26

References

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28

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CHAPTER 2

LOCALIZATION OF l-AMINOCYCLOPROPANE-l-CARBOXYLATE
OXIDASE FROM TOMATO IN TRANSGENIC YEAST

30

31
Abstract

The localization of the ethylene-forming enzyme, ACC oxidase, was studied in yeast that
had been transformed to express ACC oxidase from tomato fruits when induced with
galactose. Since at least part of the ethylene-forming activity in plants was found in
association with vacuoles, we were particularly interested to know whether ACC oxidase
would be targeted to the yeast vacuole. Transgenic ACC oxidase began to accumulate ca.
2 h after transfer of yeast cells to medium containing galactose, as was determined by
measuring ACC oxidase activity and by immunoblot analysis using ACC oxidase-specific
antibodies. Protoplasts prepared from transgenic yeast retained 84% of the ACC oxidase
activity of intact cells. Vacuoles isolated from such protoplasts did not possess ACC
oxidase activity. Immunoblot analysis confirmed that intact cells and protoplasts
contained approximately equal amounts of ACC oxidase whereas vacuoles did not contain
any detectable amounts. Differential centrifugation indicated that ACC oxidase from
transgenic yeast was associated with the particulate fiaction of the homogenate. It is not
known whether this represents specific binding of the enzyme or nonspecific attachment

that may have occurred during homogenization.

32
Introduction

The identity of l-aminocyclopropane-l-carboxylate (ACC) oxidase, the enzyme
that converts ACC to ethylene, remained elusive until recently. The breakthrough in
identifying ACC oxidase came from experiments where the antisense mRNA of a
ripening-related cDN A clone, pTOMl3, was expressed in tomato plants (Hamilton et al. ,
1990). These transgenic tomato plants exhibited greatly reduced capacity to form
ethylene from added ACC. Functional expression of the putative ACC oxidase cDNA
in yeast (Hamilton et al., 1991) and Xenopus oocytes (Spanu et al., 1991) provided
conclusive evidence that it indeed encoded the ethylene-forming enzyme. Results of
earlier work indicated that ACC oxidase was associated, at least in part, with the central
vacuole (Erdmann et al., 1989; Guy and Kende, 1984) and that the activity of the
enzyme required an intact membrane (Mayne and Kende, 1986). However, the deduced
amino acid sequence of ACC oxidase does not show a vacuolar targeting signal or
membrane association site. Also, in vitro, ACC oxidase activity was found in the soluble
fraction of tissue homogenates (Ververidis and John, 1991). In the work below, a tomato
cDNA with sequence identical to the insert of pTOM13 was expressed in yeast. ACC
oxidase activity was localized by cell fractionation with the aim of determining whether

the enzyme was targeted to the vacuole.

33
*Materials and Methods

Cloning and sequencing of cDNA

Non-degenerate oligonucleotide primers described in Chapter 1 were used to
amplify by the polymerase chain reaction a 442-bp fragment from a Lambda Zap 11
cDNA library (Stratagene, La Jolla, CA, USA) which had been constructed from
poly(A)+ RNA of ripe, unwounded tomato fruits (Lycopersicon esculentum Mill. , cv.
UC82B). This fragment was used to isolate from the above library a full-length cDNA

clone encoding ACC oxidase, which was subcloned into pUC19 for sequencing.

Production and culture conditions of transgenic yeast

Transgenic yeast containing a tomato ACC oxidase was produced as described in
Chapter 1. For the isolation of vacuoles, yeast was grown as described in Chapter 1.
For the time course of induction and differential centrifugation experiments, the yeast
was grown to mid-log phase in medium containing 2% glucose instead of galactose,
collected by centrifugation at 1000xg, and washed twice with sorbitol medium [0.6 M
sorbitol, 5 mM Tris-piperazine—N,N ’-bis(2-ethanesulfonic acid) (PIPES), pH 6.8]. The

yeast was then resuspended in medium containing 2% (+)-galactose.

Assays of A CC oxidase activity

ACC oxidase activity was measured as in Chapter 1.

34

Isolation of protoplasts and vacuoles

Protoplasts and vacuoles were isolated as described by Boller et al. (1989) with
minor modifications. Yeast cells were incubated in cell wall-digesting solution for 60
min instead of 90 min. The concentration of diethylaminoethyl-dextran (DEAE-dextran)
was lowered to 5 mg/mL to achieve better mixing with the protoplasts. Dextran sulfate

was not found to be necessary to protect the vacuoles. from lysis. The vacuoles were

collected from the interface above the 2.5% Ficoll layer.

Assays of marker enzymes

a-Mannosidase and carboxypeptidase Y assays were performed following the

procedures of Opheim (1978) and of Hemmings et al. (1981), respectively.

Protein extraction

To analyze the time course of induction, protein was extracted as in Chapter 1.
For differential centrifugation, lysed cells were centrifuged at 100xg to remove cellular
debris (Pl). A portion of the supernatant (150 uL) was centrifuged at 18,500xg for 15
min at 4°C, yielding a pellet (P2) and supernatant. This supernatant was removed to an
airfuge cylinder (Beckman, Palo Alto, CA, USA) and centrifuged at 100,000xg for 30
min in a 18° A-100 rotor. This pellet (P3) as well as P2 were resuspended in 150 ”L
of extraction buffer. The final supernatant was also brought to 150 uL. Ninety uL of

each sample were combined with 30 uL of 4x sample buffer and boiled for 10 min.

35
Isolated protoplasts and vacuoles were resuspended in an equal volume of 2x sample

buffer and boiled.

Gel electrophoresis and immunoblotting

Gel electrophoresis and immunoblotting were performed as described in Chapter
1 except for development of the blot which was done with horseradish peroxidase

conjugated to the secondary antibody (Hawkes, 1982).

Digestion with endo-fl-N-acetylglucosaminidase H (Endo-H)

Digestion of proteins with Endo-H followed a modified procedure of Trimble and
Maley (1984). Fifty uL of total protein extract from yeast induced for 5 h was
precipitated with acetone and resuspended in 20 uL of Endo-H reaction buffer (50 mM
Na-citrate, pH 5.5, 100 mM NaCl, 1 mM PMSF). Samples were digested with 5
mUnits Endo—H (Sigma Chemical Co., St. Louis, MO, USA) for 15 h at 37°C before

addition of 8 uL 4x sample buffer and boiling for 10 min.

36
Results

From a comparison of nucleotide sequences of pTOMl3 from tomato (Holdsworth
et al. , 1987) and a ripening-related gene from avocado (McGarvey et al., 1990), two
conserved regions were selected to synthesize two non—degenerate oligonucleotide primers
common to both. These primers were used to amplify by PCR a 442-bp fragment from
a tomato cDNA library prepared from poly(A)+ RNA of ripe, unwounded tomato fruit.
This fragment was subcloned into pUCl9 to yield pTEl. Partial sequencing of pTEl
confirmed 100% identity with the corresponding region of pTOM13. The pTEl insert
was used to screen at moderately high stringency (final wash = 0.5x SSC, 65°C)
approximately 50,000 primary plaques of the same cDNA library for a full-length clone.
Five strongly hybridizing plaques were selected. One of these, pTE2, was chosen for
sequencing based on its insert size. Sequencing of the ends of the clone confirmed that
it contained the correct 5’ and 3’ coding sequence and, therefore, was assumed to be full-
length. With the exception of two unreadable base pairs, the sequence of pTE2 was
identitical to the corrected sequence of pTOMl3 (Hamilton et al. , 1991).

To determine whether the insert encoded a functional ACC-oxidase, we subcloned
the pTE2 insert into pYES2.0, a yeast expression vector with a galactose-inducible
promoter, to yield pYTE2. We then transformed yeast strain F808 with pYTE2 to create
TF808. Of the three transformants tested, all were able to convert ACC to ethylene.

One of the transformants, TF808-1, was selected for further investigation.

37

When transferred to inductive conditions, ACC-oxidase activity began to increase
in TF808-1 cells after about 2 h. Activity continued to increase for the duration of the
experiment (Figure 2.1 A). Immunoblot detection showed that the appearance and
accumulation of the transgenic ACC oxidase (M, 36 kDa) followed the pattern seen in
the activity assay during the first 4 h (Figure 2.1 B). TF808-1 grown in noninducing
glucose medium did not produce ethylene from ACC above background levels and did
not accumulate antigenic protein (data not shown). Yeast transformed with the vector
alone was also unable to produce ACC oxidase, even under inductive conditions (data
not shown). The antibody also recognized a protein of 36 kDa in extracts from elicitor-
treated tomato cell cultures (Figure 2.1 B, lane L). It had been shown previously that

elicitor—treated tomato cells expressed high levels of ACC oxidase mRNA and displayed

a high activity of the enzyme (Spanu et al., 1991).

 

38

Legend for Figure 2.1. (A) Immunoblot analysis of protein extracts from yeast cells
induced with galactose for 0 to 5 h (lanes 0-5; each lane represents the extract from an
equal number of cells) and from tomato cell cultures treated with yeast-derived elicitor
for 4 h (lane L). (B) Ethylene synthesis in transformed yeast cells incubated with 1 mM
ACC, 30 mM ascorbate, and 0.1 mM FeSO,.

 

 

39

 

 

 

 

 

 

 

A kDa
4106
4 80
< 49.5
’—-—- " ‘ 32.5
4 27.5
0 1 2 2.5 3 4 5
TlMElhl
3 12
E 10 - a -
8 8 ~ -
0'6
c
if? 6 + -
'5- /
In: 4 _ 4
3 D
s 2 - a/ -
.9.- n’
0 ...—Egan, L
0 1 2 3 6
Timelh)

 

Figure 2.1. Time course of induction of ACC oxidase in transgenic yeast (TF-808-1)

following transfer to galactose medium.

 

 

 

40

The localization of ACC oxidase activity in transgenic yeast was examined to
determine if it was present in the vacuole. While protoplasts retained 84 % of the ACC
oxidase activity found in intact cells, isolated vacuoles were unable to convert ACC to
ethylene (Table 2.1). Microscopic inspection was used after each assay to verify that the
loss of activity did not result from lysis of vacuoles or protoplasts. The results of the
ACC oxidase activity assays were confirmed by immunoblot detection (Figure 2.2). An
aliquot from each of the samples used in the activity assays was loaded on an SDS-
polyacrylamide gel such that each lane represented an equal number of cells, protoplasts,
and vacuoles, as determined both by measuring a-mannosidase activity and by counting
under a microscope. Whole cells and protoplasts contained similar amounts of ACC

oxidase protein, but none could be detected in vacuoles. Carboxypeptidase Y assays

confirmed that the contents of the vacuolar lumen were still present. Thus, the lack of
immunologically reactive protein in the vacuolar fraction could not have resulted from
loss of vacuolar components during the isolation procedure. Also, comparison of freshly
isolated protoplasts and protoplasts kept on ice for 6 h showed that the protein had not

been degraded, even after periods well in excess of the time required for isolation of the

vacuoles (Figure 2.2, lane 3).

 

 

41

Table 2.1. Ethylene-forming activities of cells, protoplasts, and vacuoles. Values
represent the means of 4 assays from 2 separate isolations.

 

Cells Protoplasts Vacuoles
Ethylene [pmol h‘l (10° cells or vacuoles)"]
0.68 i 0.06 0.57 i 0.08 <0.01

 

 

42

 

kDa 1 2 '3 4
106 >
80.0»

49.5 v

32.5 r

27.5 >

 

 

 

Figure 2.2. Immunoblot analysis of protein extracts from yeast cells, protoplasts, and
vacuoles. Lanes represent equal number of cells (lane 1), protoplasts (lane 2),
protoplasts stored on ice for 6 h (lane 3), and vacuoles (lane 4) from cells that had been
induced with galactose for 24 h.

43

Upon differential centrifugation, the transgenic ACC oxidase was recovered in the
particulate (PZ) fraction (Figure 2.3, lane 6). The microsomal fraction (P3) and the
soluble fraction contained little or no ACC oxidase (Figure 2.3, lanes 7 and 8,
respectively). The higher molecular weight bands seen on the blot were present in the
uninduced yeast (lanes 1-3), suggesting cross-reactivity with yeast proteins. Samples
treated with Endo-H (Figure 2.3, lane 9) and the respective control not treated with
Endo-H (lane 10) had the same molecular mass, indicating that ACC oxidase is not

glycosylated.

 

kDa 1 2 3 4 5 6 7 8
80.0»

46.5 > ........ E

32.5 b
27.5 D

 

 

 

Figure 2.3. Immunoblot analysis of proteins from differential centrifugation and from
Endo-H treatment. Lanes 1 to 3, 0-h induction; lanes 4 to 6, 5-h induction with
galactose; lanes 1 and 4, 18,500 x g pellet (P2); lanes 2 and 5, 100,000 x g pellet (P3);
lanes 3 and 6, soluble fraction. Each fraction was brought to the same volume (150 uL)
of which aliquots were used for analysis (see Materials and Methods). Lanes 7 and 8,
immunoblot analysis of total protein extracted from cells induced for 5 h and incubated
with (lane 7) and without (lane 8) Endo-H.

45

Discussion

For many years, ACC oxidase remained the elusive enzyme in the ethylene
biosynthetic pathway. A large body of evidence suppOrted the notion that the conversion
of ACC to ethylene required membrane integrity. Osmotic and cold shock and treatment
with detergents inhibited oxidation of ACC to ethylene (Apelbaum et al., 1981a) as did
ionophores (Apelbaum et al., 1981b; Mayne and Kende, 1986; Yu et al., 1980).
Vacuoles isolated from pea leaves contained 80% of the ethylene-evolving capacity of
protoplasts from which the vacuoles had been prepared (Guy and Kende, 1984). The
ACC-oxidase activity in the vacuole displayed the same stereospecificity as exhibited by
the enzyme in vivo. The activity of the enzyme was lost upon lysis of the vacuole,
leading to the conclusion that membrane integrity was required for the functioning of the
ethylene-forming enzyme (Mayne and Kende, 1986). However, recent results based on
analysis of cDNA clones encoding ACC oxidase do not support vacuolar localization of
ACC oxidase or requirement for membrane integrity. The cDNA-derived amino acid
sequence of ACC oxidase from tomato (Hamilton et al., 1991; Spanu et al., 1991) and
that of putative ACC oxidase from avocado fruits (McGarvey et al. , 1990) and carnation
flowers (Wang and Woodson, 1991) do not indicate targeting sequences or domains for
association with a membrane. Also, the reconstituted in vitro activity of ACC oxidase
from melon fruits proved to be soluble (Ververidis and John, 1991).

The localization of ACC oxidase in transgenic yeast was examined mainly to

determine whether at least part of the ethylene-forming activity was associated with the

46

vacuole. Previous work has shown that certain plant proteins targeted to the vacuole by
way of the endomembrane system are also properly transported to the vacuole when
expressed in yeast cells (Chrispeels, 1991). In addition, there is a possibility that
cytoplasmic proteins lacking a conventional signal sequence may be imported directly
into the yeast vacuole (Chiang and Schekman, 1991).- The results shown in Table 2.1
and Figure 2.2 demonstrate conclusively that ACC oxidase is not present in the yeast
vacuole. Because the vacuole can act as a lysosome-like compartment, the possibility
exists that a portion of the protein may have been targeted to the vacuole and rapidly
degraded. High levels of ACC oxidase activity, however, were measured in cells and
protoplasts without detecting the protein in the vacuole. Thus, the tomato fruit ACC
oxidase does not require a vacuolar localization in order to be functional in yeast.

While the tomato protein does not contain a recognizable targeting signal for the
endoplasmic reticulum, there is a potential glycosylation site (Asn-X-Ser) at amino acids
335-337. To determine whether the protein was glycosylated, the protein extact from
induced cells was incubated with Endo-H, an enzyme that hydrolyzes N-linked high
mannose oligosaccharides. As shown in Figure 2.3 (lanes 9 and 10) there is no
difference in molecular size between the Endo-H-treated and untreated proteins.

The results from differential centrifugation show that ACC-oxidase is associated
with a particulate fraction. We obtained identical results regardless of whether the
fractionation was performed 2 or 24 h after the induction of expression. Thus,
association with a membrane fraction does not appear to be the result of overexpression

of the protein. It is not yet known whether it represents specific binding of the enzyme

47

to a particular cell component or nonspecific attachment that may have occurred during
homogenization.

The consistent association of ACC oxidase with the particulate fraction raised the
possibility of protein-protein interactions between ACC oxidase and an integral membrane
protein. A predicted secondary structure for ACC Oxidase from tomato fruits was
obtained by a modified Chou-Fossman analysis from sequence analysis programs MCF
and Amphi (available from AR. Crofts, University Of Illinois, Urbana, IL, USA). A
predicted amphipathic helix extends from Met120 to Glu140 and possibly extending to
Lys150 (see consensus ACC oxidase sequences, Figure 1.3). Beginning with Phel23,
which is known to be able to substitute for leucine residues in leucine zippers without the
loss of function (Gentz et al., 1989), a leucine residue repeats every seven amino acids,
a characteristic of a leucine zipper (Landschultz et al., 1988). While an amphipathic
helix/leucine zipper motif is present in all ACC oxidases, it is also present in a similar
region of 2-oxoglutarate-dependent dioxygenases from plants (Figure 1.4). Crystal
structure data from a related fungal enzyme, isopenicillin N synthase, indicate that the
amphipathic helix is a structural feature involved in stabilizing the B-sheets that form the
active site region (Roach et al., 1995). Thus, the helix is more likely to fill a structural
role in ACC oxidases than to be involved in protein-protein interactions. The association
of ACC oxidase with the particulate fraction appears to be from nonspecific attachment

that occurred during homogenization.

References

Apelbaum A, Burgoon AC, Anderson JD, Solomos T, and Lieberman M (1981)
Some characteristics of the system converting l-aminocyclopropane-l-carboxylic
acid to ethylene. Plant Physiol 67: 80-84

Apelbaum A, Wang SY, Burgoon AC, Baker JE, and Lieberman M (1981) Inhibition
of the conversion of l-aminocyclopropane-l-carboxylic acid to ethylene by
structural analogs, inhibitors of electron transfer, uncouplers of oxidative
phosphorylation, and free radical scavengers. Plant Physiol 67: 74-79

Boiler T, Diirr M, and Wiemken A (1989) Transport in isolated yeast vacuoles:
Characterization of arginine permease. Methods Enzymol 174: 504-518

Burnette WN (1981) "Western blotting: electrophoretic transfer of proteins from sodium
dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic
detection with antibody and radioiodinated Protein A. Anal Biochem 112: 195-203

Chiang H-L, Schekman R (1991) Regulated import and degredation of a cytosolic
protein in the yeast vacuole. Nature 350: 313-318

Chrispeels M (1991) Sorting of proteins in the secretory pathway. Annu Rev P l a nt
Physiol Mol Biol 42: 21-53

Erdmann H, Griesbach RJ, Lawson RH, Mattoo AK (1989) l—Aminocyclopropane—l-
carboxylic-acid-dependent ethylene production during re-formation of vacuoles in
evacuolated protoplasts of Petunia hybrida. Planta 179: 196-202

Gentz RF, Rauscher III F], Abate C, Curran T (1989) Parallel association of Fos and
Jun leucine zippers juxtaposes DNA binding domains. Science 243: 1695-1699

Guy M, Kende H (1984) Conversion of l-aminocyclopropane-l-carboxylic acid to
ethylene by isolated vacuoles of Pisum sativum L. Planta 160: 281-287

Hamilton AJ , Bouzayen M, Grierson D (1991) Identification of a tomato gene for the

ethylene-forming enzyme by expression in yeast. Proc Natl Acad Sci USA 88:
7434-7437

Hamilton AJ, Lycett GW, Grierson D (1990) Antisense gene that inhibits synthesis of
the hormone ethylene in transgenic plants. Nature 346: 284-287

49

Hawkes R (1982) Identification of concanavalin A-binding proteins after sodium dodecyl
sulfate-gel electrophoresis and protein blotting. Anal Biochem 123: 143-146

Hemmings BA, Zubenko GS, Hasilik A, Jones EW (1981) Mutant defective in
processing of an enzyme located in the lysozome-like vacuole of Saccharomyces
cerevisae. Proc Nat Acad Sci USA 78: 435-439

Holdsworth MJ, Bird CB, Ray J, Schuch w, and’Grierson D (1987) Structure and
expression of an ethylene-related mRNA from tomato. Nucleic Acids Res 15:
731-739

Laemmli UK (1970) Cleavage of structural proteins during assembly of the head of
bacteriophage T4. Nature 227: 680-685

Landschulz WH, Johnson PF, McKnight SL (1988) The leucine zipper: A Hypotheti-
cal structure common to a new class of DNA binding proteins. Science 240:
1759-1764

Mayne RG, Kende H (1986) Ethylene biosynthesis in isolated vacuoles of Vicia faba
L. - requirement for membrane integrity. Planta 167: 159-165

McGarvey DJ, Yu H, Christofferson RE (1990) Nucleotide sequence of a ripening-
related cDNA from avacado fruit. Plant Mol Biol 15: 165-167

Opheim DJ (1978) a-D-Mannosidase of Saccharomyces cerevisiae characterization and
modulation of activity. Biochem Biophys Acta 524: 121-130

Roach PL, Clifton IJ, Fulop V, Harlos K, Barton GJ, Hajdu J, Andersson I,
Schofield CJ, Baldwin JE (1995) Crystal structure of isopenicillin N synthase
is the first from a new structural family of enzymes. Nature 375: 700-704

Spanu P, Reinhardt D, Boller T (1991) Analysis and cloning of the ethylene-
forming enzyme from tomato by functional expression of its mRNA in Xenopus
laevis oocytes. EMBO J 10: 2007-2013

Trimble RB, Maley F (1984) Optimizing hydrolysis of N-linked high mannose oligo-
saccharides by Endo-B-N-acetylglucosaminidase H. Anal Biochem 141: 515-522

Ververidis P, John P (1991) Complete recovery in vitro of ethylene-forming enzyme
acitivity. Phytochemistry 30: 725-727

Wang H, Woodson WR (1991) A flower senescence-related mRNA from carnation
shares sequence similarity with fruit ripening-related mRNAs involved in ethylene
biosynthesis. Plant Physiol 96: 1000-1001

50

Yu Y-B, Adams DO, Yang SF (1980) Inhibition of ethylene production by 2,4-
dinitrophenol and high temperature. Plant Physiol 66: 286-290

CHAPTER 3

SEQUENTIAL INDUCTION OF THE ETHYLENE BIOSYNTHETIC
ENZYNIES BY INDOLE-3-ACETIC ACID IN ETIOLATED PEAS

51

52
Abstract

Ethylene induced an increase in the accumulation of l-aminocyclopropane-l-carboxylate
(ACC) oxidase transcript level and enzyme activity in the first intemode of 5- to 6-day-
old etiolated pea (Pisum sativum L.) seedlings. Indole-3-acetic acid (IAA), which
stimulates ethylene production by enhancing ACC Synthase activity, also caused an
increase in ACC oxidase transcript and activity levels. The IAA-induced increase in
ACC oxidase mRNA level and enzyme activity was blocked by 2,5-norbomadiene
(NBD), a competitive inhibitor of ethylene action. This indicates that IAA induces ACC
oxidase through the action of ethylene. The level of ACC synthase mRNA and enzyme
activity started to increase less than 1 h after the start of IAA treatment, whereas ACC
oxidase activity and transcript levels began to rise after 2 h of IAA treatment. These
results indicate that the enzymes of ethylene biosynthesis are sequentially induced after
treatment of intact pea seedlings with IAA. The increase in ACC synthase activity leads
to the production of ACC, which is converted by the low constitutive level of ACC
oxidase activity to ethylene. Ethylene promotes the accumulation of ACC oxidase

mRNA and the increase in ACC oxidase activity through a positive fwdback loop.

53
Introduction

Ethylene is a gaseous hormone that regulates a wide range of physiological
responses in vegetative tissues (Abeles et al., 1992). In etiolated pea seedlings, an
asymmetric distribution of ethylene biosynthesis may be involved in the asymmetry of
growth that leads to the formation of the apical hook (Schierle and Schwark, 1988). This
asymmetry in ethylene production may result from a gradient in auxin concentration
between the outer and inner portions of the apical hook. Ethylene production in
vegetative tissues is thought to be regulated by the level of free IAA (Yang and Hoffman,
1984). While it is difficult to establish the exact auxin concentration at its site of action,
experiments with 3H-IAA show that the IAA level within the interior portion of the hook
is approximately 4-fold greater than in the outer portion of the hook (Schwark and
Schierle, 1992). Although auxin is normally associated with cell elongation, higher
concentrations (3 uM to 1 mM) of IAA inhibit growth by stimulating ethylene production
(Burg and Burg, 1966). Thus, a high local concentration of IAA in the inner portion of
the hook may be responsible for the increased ethylene production in this region (Schierle
and Schwark, 1988) and this may, ultimately, lead to inhibition of growth. Studying the
effects of IAA on the enzymes of ethylene biosynthesis will help to understand how these
hormones interact in this response.

Ethylene production is normally low in vegetative tissues but can be increased by
a variety of stimuli, including wounding and IAA (Abeles et al., 1992). In most cases,

the increase in ethylene production appears to result from an increase in the transcript

54

and activity levels of ACC synthase, the first committed enzyme of ethylene biosynthesis
(Kende, 1993). ACC synthase catalyzes the formation of ACC from S—adenosyl—L-
methionine (AdoMet). ACC is converted to ethylene by the second enzyme, ACC
oxidase, which is thought to be constitutively present in most vegetative tissues (Yang
and Hoffmann, 1984). Because a stimulus that promotes ethylene production usually
causes ACC synthase activity to increase rapidly from extremely low or undetectable
levels, and because ACC oxidase is usually constitutively present at least at low levels,
ACC synthase has been widely regarded as the rate-limiting step in ethylene biosynthesis
(Yang and Hoffman, 1984). There is evidence, however, that changes in ACC oxidase
levels may also contribute to the overall increase in rates of ethylene production. Using
in vivo assays, it has been shown that ethylene pretreatment increases ACC oxidase
activity in citrus leaves (Riov and Yang, 1982), etiolated pea seedlings (Schierle et al.,
1989), and carnation petals (Drory et al., 1993), and wounding of ripening tomato fruit
induces the accumulation of ACC oxidase transcript levels (Holdsworth et al., 1987).
Wounding also causes an increase in ACC oxidase activity levels in winter squash
mesocarp (Hyodo et al., 1993) and in ACC oxidase transcript and activity levels in
etiolated mung bean hypocotyls (Kim and Yang, 1994). In these cases, wound-induced
accumulation of ACC oxidase could be blocked by treatment with NBD, a competitive
inhibitor of ethylene action (Sisler and Yang, 1984). In orchid flowers, pollination
caused an increase in ACC oxidase transcript levels, and this increase could be inhibited
by NBD (O’Neill et al., 1993). These results show that a stimulus causing an increase

in ethylene production can also cause an increase in ACC oxidase activity. It has also

55
been suggested that the enzymes of ethylene biosynthesis are sequentially induced by a

stimulus promoting ethylene production (Hyodo et al., 1993). It is currently not known
whether, in these instances, ACC oxidase is a rate-limiting enzyme in the production of
ethylene.

The work below reports on the effects of IAA on ACC oxidase transcript and
activity levels. The timing Of the induction of both ACC synthase and ACC oxidase is
also compared. These studies use the first intemode of 5- to 6-day-old etiolated peas
because this tissue has a very low basal level of ethylene production (Schierle et al. ,
1989), making it easy to detect changes in ACC synthase and ACC oxidase mRNA and

enzyme activity levels.

56
Materials and methods

Plant material

Pea Ms (Pisum sativum L., cv. Alaska; Cliston Seed Co., Faison, NC) were
imbibed overnight in aerated tap water. Seedlings were grown in vermiculite at 25°C
in darkness. In all experiments, treatments were performed on intact, 5- to 6-day-old
seedlings that had formed a third node, and the start of the treatments was designated as
0 h. At the times indicated, 2-cm sections were isolated from the Mlings and
immediately frozen in liquid nitrogen. Tissue from the same experiment was used for
both the enzyme assays and RNA isolations. All manipulations were performed under

safe green light (530-590 nm).

Hormone and inhibitor treatment of plant material

For ethylene treatments, mdlings were enclosed in 9-L desiccators fitted with
injector ports that were sealed with rubber serum vial caps. Ethylene was injected into
the desiccators, and the proper internal ethylene concentration was confirmed by gas
chromatography. Purafil (Purafil, lnc., Norcross, GA), which absorbs ethylene, was
added to control treatments in a dish at the bottom of the desiccator to prevent
accumulation of ethylene produced by the seedlings. IAA solutions contained 100 pM
IAA, 0.05% ethanol, and 0.05% Tween-20, adjusted to pH 6.0 with dilute NaOH.
Control solutions of the same pH were identical except that IAA was omitted. To disturb

the seedlings as little as possible, the solutions were applied to the intact plants as a fine

57

mist using a spray bottle. Treatments with NBD were performed in 9-L desiccators.
Immediately after being treated with IAA or control solutions, the seedlings were placed
in the chamber and NBD was injected to yield a concentration of 3000 uL/L in the gas

phase.

Measuring the rate of ethylene production

At the indicated times after treatments, 7 to 10' sections were isolated from the
first intemode and enclosed in 4-mL tubes fitted with serum vial caps. After 30 min, 1
mL of headspace was withdrawn for determination of ethylene concentration by gas
chromotography. Because the lag time for wound-ethylene production in etiolated pea
stems is approximately 26 min (Saltveit and Dilley, 1978), taking the measurements after

30 min minimizes any contributions of wound effects to the ethylene production rates.

ACC oxidase enzyme assays

The ACC oxidase assay was modified from that of Ververidis and John (1991).
For the experiments with IAA treatment, weighed, frozen stem sections were ground in
liquid nitrogen. An extraction buffer (1 mL/g FW) consisting of 100 mM Tris-HCl, pH
7.2, 30 mM Na-ascorbate, and 10% glycerol was added to the frozen powder which was A
allowed to thaw to a slurry. The total extract was transferred to a cold, 1.5-mL
microcentrifuge tube on ice and centrifuged at 15,000 g for 10 min at 4°C. The
supernatant was used directly in the assay. Neither polyvinylpolypyrrolidone nor Triton

X-100 were necessary in the extraction buffer to recover maximum activity from the stem

58

sections, as was reported for apple fruits (Dong et al., 1992). For the time course of
induction by ethylene, the addition of 1 mM ACC to the extraction buffer was necessary
to recover maximum enzyme activity. The addition of ACC to the extraction buffer for
the IAA-treated tissue did not increase the recoverable ACC oxidase activity. A likely
explanation for this observation is that the ACC stabilized the enzyme during extraction
and that IAA-treated tissue produced sufficient amounts of ACC to stabilize ACC oxidase
without the addition of ACC to the extraction buffer.

The activity assays consisted of 200 pL extraction buffer, 50 uL of 40 mM ACC,
50 uL of 2 mM FeSO4, and 1.7 mL of the enzyme extract in a total volume of 2 mL.
The reaction mixtures were shaken at 30°C for l h in 9-mL screwcap tubes, each fitted
with a teflon-coated septum (Fischer Scientific, Pittsburgh, PA). At the end of this time
period, 1 mL of the headspace was removed and analyzed by gas chromatography. The
in vitro activity was saturated at 1 mM ACC, required ascorbate and FeSO, for maximal
activity, was inhibited by Co“, and was completely abolished by 5-min treatment at
90°C. These results agree with previously established parameters for specific ACC

oxidase activity.

ACC synthase enzyme assays

Because of the low ACC synthase activity recovered from pea-stem tissue, it was
necessary to modify the extraction procedure from that described by Jones and Kende
(1979). Approximately 10 g FW of stem tissue was ground in liquid nitrogen and

extracted with 0.5 mL/ g FW of buffer (200 mM Na-phosphate, pH 8.0, 5 mM DTI‘, 10

59
14M pyridoxal 5-phosphate). The slurry was centrifuged at 25,000 g for 20 min at 4°C.

The supernatant was dialyzed overnight against two changes of buffer (10 mM Na-
phosphate, pH 8.0, 5 uM pyridoxal 5-phosphate) at 4°C. The volume of the dialyzed
extract was reduced by placing the dialysis bag onto dry polyethylene glycol until 1 mL
Of extract represented approximately 1 g FW of tissue. In the assay, 2 mL of
concentrated extract was combined with 200 pL of 1 M Na-phosphate, pH 8.0, and
either 50 ul of 0.2 M AdoMet or H20 for a total volume of 2.25 mL in a 9-mL tube, and
the reaction mixture was shaken at 30°C for 1 h. The ACC produced was determined

by the method of Lizada and Yang (1979).

Cloning of ACC synthase

To clone the gene for IAA-induced ACC synthase, reverse transcription (RT)-
polymerase chain reaction (PCR) was performed using total RNA isolated from tissue
treated for 2 h with IAA. In the RT reaction, 2 uL of 0.2 ug/pL oligo(dT),, (Pharmacia,
Piscataway, NJ), 10.6 ”L of 2.5 mM dNTP mixture, 0.4 uL of 40 U/uL RNAsin RNAse
inhibitor (Promega, Madison, WI), 1 pL of 200 U/uL M-MLV reverse transcriptase with
4 uL of the supplied 5X RT buffer (Promega), and 2 11L of l ug/uL total RNA were
combined for a total volume of 20 uL. This mixture was incubated at 37°C for 1 h, and
the reaction stopped by incubation at 95°C for 10 min. Two uL of the RT reaction was
used for PCR. Two degenerate oliogonucleotide primers, 5’-CTC(GAATTC)
ACCAAYCCNTCNAAYCCNYTRGG-3’and5’-CTC(AAGCTI')ACNARNCCRAARC-

TYGACAT-B’ based on conserved amino acid sequences (TNPSNPLG and MSSFGLV,

60

respectively) were synthesized containing EcoRI and HindIII restriction sites (shown in
parentheses) at the 5’-ends, respectively, and used for PCR. Thermocycling was
performed at 94°C for 1 min, 42°C for 1 min, and 72°C for 1 min, for 35 cycles. The
products were digested with EcoRI and HindIII, separated by agarose gel electrophoresis,
recovered by electrophoresis onto DE-8l paper (Sambrook et al. , 1989), ligated into
pBluescript SK- (Stratagene, La Jolla, CA), and transformed into INVaF’ cells
(Invitrogen, San Diego, CA). Five independent clones were selected for sequencing.
The sequences of all five were identical in both strands except for the primer regions.
One of these clones (317 bp) was selected for further study. Since a different ACC
synthase cDNA clone (PS-ACSl, see Chapter 4) had been previously isolated from the
hook region of etiolated pea seedlings, the PCR-generated clone was designated PS-ACS2

(see Figure 3.1).

RNA blot analysis

All RNA isolations were performed as described by Puissant and Houdeline
(1990). For ACC oxidase RNA blots, 20 pg of total RNA was separated on a 1.2%
agarose-formaldehyde gel. Ethidium bromide staining of the ribosomal bands was used
to confirm equal loading of lanes. Gels were transferred to Hybond-N nylon membranes
(Amersham, Arlington Heights, IL). The filters were baked at 80°C for 1-2 h and
prehybridized at 62 °C with 5X SSPE, 10X Denhardt’s, 0.1% SDS, 0.25 mg/mL salmon
sperm DNA, and 50% formamide, for at least 4 h. A full-length (1122 bp) pea ACC

oxidase clone (pPE8) (Peck et al., 1993) in pBluescript SK- (Stratagene) was used to

61
synthesize a ”P-labeled antisense strand RNA probe by using T7 RNA polymerase.

Hybridization was performed at 62°C in the prehybridization buffer. The filters were
washed 3 times for 15 min each in 0.2x SSC, 0.2% SDS, at 62°C. Autoradiography
was performed using Hyperfilm-MP (Amersham) at -80°C with two amplification
screens. The signals were quantified with a phosphorimager (Molecular Dynamics,
Sunnyvale, CA).

For ACC synthase RNA blots, conditions were as described except that 5 ug of
poly(A)+ RNA was used. The poly(A)" RNA was isolated using the Mini-Oligo(dT)
Cellulose Spin Column kit (5 Prime -> 3 Prime, Inc., Boulder, CO) according to the
manufacturer’s instructions. The riboprobe was prepared from PS-ACSZ, a 317-bp PCR

product.

62

1 ACTGTTATGGACAGAAACACACTAAGAACTGTCATCACTTTCATCAACGAAAAGCGTATC
T V M D R N T L R T V I T F I N E K R I

61 CAATCTAATAAGCGACGAAATCTACGCTGCAACGGTTTTTAGCCACCAAGTTTCATAAGC
H L I S D E I Y A A T V F S H P S F I S

121 ATAGCGGAGATAATCGAACATGACACAGACATTGAATGCGACCGTAACCTCGTTCACATA
I A E I I E H D T D I E C D R N L V H I

181 GTTTATAGTCTTTCAAAAGTCATGGGATTCCCGGGGTTTAGAGTTGGTATAATATACTCA
VY§LSKDMGrFP§FRV§IIYS

241 TACAACGACACCGTTGTTGATTGCACGCGCAAA
Y N D T V V D C T R K

Figure 3.1. Nucleotide and deduced amino acid sequence of ACC synthase cDN A clone
PS-ACS2. The sequence does not contain the primers used for PCR (described in
Materials and Methods). The conserved active site region is underlined.

63
Results

Cloning of an IAA-inducible ACC synthase

To obtain a probe for measuring ACC synthase transcript levels, RT—PCR was
used to clone an ACC synthase cDNA fragment (PS-ACS2) from the first intemode of
etiolated pea seedlings treated for 2 h with IAA. This cDNA contained the nucleotide
sequence encoding the active site of ACC synthase. PS-ACS2 (Figure 3.1) has 79% and
67% nucleotide identity with the corresponding regions of two IAA—induced ACC
synthase cDNA clones from etiolated mung bean hypocotyl sections (Botella et al. , 1992;

Kim et al., 1992).

Ethylene increases ACC oxidase activity and transcript levels

Schierle et al. (1989) found that ethylene pretreatment of stem sections from
etiolated pea seedlings stimulated in vivo ACC oxidase activity. The present work
expanded upon the previous experiments by using intact seedlings for treatments and an
in vitro enzyme assay. ACC oxidase transcript levels and enzyme activity increased with
the duration of exposure to 40 pL/L ethylene (Figure 3.2). ACC oxidase transcript and
enzyme activity were present at low but detectable levels in the untreated tissues. After
12 h of exposure to ethylene, the transcript level had increased almost 100-fold, and the
enzyme activity had increased about 10-fold. The changes measured in in vitro enzyme

assays were consistent with those seen in vivo when the conversion of exogenous ACC

(1 mM)

A 046681212(h)
Ethylene - + + - + + -

 

 

  

 

   

 

45-
4 -
E o
.5? 35—
2-
03,, 30—
%: 25L
--z"
X N _
0" _
2515
’° w;
5

 

0 2 4 6 8 10 12

Duration of Treatment (h)

Figure 3.2. Effect of ethylene on ACC oxidase transcript abundance (A) and enzyme
activity (B). At 0 h, intact seedlings were placed in desiccators with (O) or without (v)
40 uL/L ethylene. At the times indicated, the seedlings were removed from the
desiccators, and 2-cm sections from the first intemode were isolated and used for RNA
isolation and enzyme assays. For RNA blot analysis (A), 20 pg of total RNA was
separated on a 1.2% agarose—formaldehyde gel, transferred to a nylon membrane, and
probed with a 32P-labelled antisense RNA strand prepared from pPE8 (Peck et al., 1993).
The experiment was performed four times with similar results.

65

to ethylene was measured in stem sections (data not shown). The increase in ACC
oxidase activity was not a result of handling during treatment or of enclosing seedlings
in desiccators because neither the transcript level nor the activity of the enzyme increased
in the control treatments (Figure 3.2). Since ethylene can cause the accumulation of
ACC synthase activity in some tissues (Yang and Hoffman, 1984), it could be argued that
ethylene induced ACC oxidase activity by increasing the availability of ACC. To address
this possibility, the levels of ACC synthase transcript, ACC synthase enzyme activity,
and ACC were measured after ethylene treatment. No changes in any of these
parameters were detected during the treatment of the seedlings with ethylene (data not

shown). Therefore, ACC oxidase activity is induced by ethylene and not by ACC.

IAA increases ACC oxidase activity and transcript levels via ethylene

Because ethylene induced the accumulation of ACC oxidase activity, a stimulus
that promotes ethylene production may also stimulate ACC oxidase activity. In the first
intemode of etiolated pea mlings, 100 uM IAA stimulated ethylene production via an
increase in extractable ACC synthase activity (Figure 3.3). A control solution of the
same pH did not cause this increase, indicating that the response is specific to auxin and
that the application method does not cause an increase in ACC synthase activity or
ethylene production. ACC oxidase transcript levels and enzyme activity increased
following treatment with IAA but not in control tissue (Figure 3.4). NBD, an inhibitor
of ethylene action, prevented the increase in ACC oxidase transcript and enzyme activity

levels after treatment with IAA (Figure 3.4). NBD did not prevent the IAA-induced

 

 

 

7 1.0
3A6l- v c
"' l —oa-9r~
'3 “- ' ' z 3
2.. l- 3 LI.

1 '0'-
01 e —08 0'0!
9 4 IAA - L
01 ll ' T
.c .c o
E _ 3L— : _04 C I
>4 0 1 ' 2 -'
i 0 . c
m 52E Ev
0v m
2 1! Control ‘0'2
'4!" v/ ’7
o l l l l l l 0.0

 

0 2 4 6 8 10 12
Duration of Treatment (h)

Figure 3.3. Effect of IAA on ACC synthase activity and rate of ethylene production in
the first intemode of etiolated pea seedlings. At 0 h, seedlings were sprayed with
solutions containing 100 pM IAA (O, v) or no IAA (O, V). At the times indicated,
2-cm sections were isolated from the first intemode and used for ACC synthase enzyme
assays (O , O) or for measuring ethylene production (v , V). The experiment was
performed twice with similar results.

67

Legend for Figure 3.4. Treatments were as described in Figure 3.3. Plants were treated
with auxin (0,0) or control solutions (v,V) in the presence (O,v) or absence (.3)
of NBD. RNA blot analysis was performed as described in Figure 3.2. The experiment
was performed four times with similar results.

 

 

o 4 6 a 12 12 h
A IAA + + + +' ( )
NBD - - + - - +
F" U H . a
B o 4 6 6 a 12 12 (h)
IAA ' ' " I I -

NBD- - - + - 3 +

 

 

   

 

 
 

C 26 .
IAA ‘
new 24 -
cei" E
>
Iprnrlri 35 20 '
3'70 16 .- /
a 1:
:2 v .
x 1.. 12 -
O O
o -
o 5 6 -
< C
4 Control ontrol
. +NBD

 

 

0 2 4 6 8 10 12
Duration of Treatment (h)

 

 

Figure 3.4. Effect of IAA and NBD on ACC oxidase transcript abundance (A,B) and
enzyme activity (C).

69

accumulation of ACC synthase activity (data not shown), showing that NBD did not
prevent the response to IAA. To determine if NBD prevented the increase in ACC
oxidase activity non-specifically, saturating levels of ethylene (300 uL/L) were added to
the IAA +NBD treatment. Ethylene restored the ACC oxidase activity and transcript
levels in the presence of NBD (Figure 3.5), supporting earlier evidence that NBD acts
specifically as a competitive inhibitor of ethylene action. The difference in transcript
abundance between the IAA and the IAA+NBD+ethylene treatments (Figure 3.5) was
not surprising. In the IAA treatment, the amount of transcript reached a maximum at
8 h and decreased by 12 h (Figure 3.4A), presumably because IAA-induced ethylene
production had ceased (note the pattern of ethylene production in Figure 3.3). In the
presence of ethylene, the ACC oxidase transcript level continued to increase during entire
period of treatment (Figure 3.2). These results show that IAA increases the levels of

ACC oxidase transcript and activity via ethylene.

70

 

    

IAA IA+A IA+A
NBD NBD
c,H,

0|
0
l

“V

.2? 40-
g...
a...“ 30-
$4:
'5 v
-;::N 20-
00
a:
<V1o-

 

 

 

 

Figure 3.5. Competitive effects of NBD and ethylene on ACC oxidase transcript
abundance and enzyme activity during IAA treatment. Swdlings were treated with auxin
as described in Figure 3.3 for 12 h with or without NBD or ethylene . RNA blot
analysis was as described in Figure 3.2. The experiment was performed twice with
similar results.

71

The evidence that IAA treatment resulted in an increase in ACC synthase activity
and that the ethylene produced caused an increase in ACC oxidase activity indicated that
the ethylene biosynthetic enzymes are sequentially induced. If so, the increase in ACC
synthase activity should be detectable before the increase in ACC oxidase activity.
Figure 3.6 shows the changes in ACC synthase and ACC oxidase transcript levels and
activities during the first 2 h of IAA treatment. The levels of ACC synthase transcript
and enzyme activity increased within the first hour (Figure 3.6, A and C). ACC oxidase
transcript, however, did not begin to increase until 2 h after the start of IAA treatment
(Figure 3.6B). ACC oxidase activity did not increase during the first 2 h (Figure 3.6C),

although it did increase about 2-fold by 4 h (Figure 3.4C).

72

Legend for Figure 3.6. Treatment conditions were as described in Figure 3.3, and RNA
blot analysis as in Figure 3.2, except that 5 pg poly(A)+ RNA was used with an ACC
synthase antisense RNA strand made from pPS-ACS2 as a probe. The experiment was
performed three times with similar results.

73

A 01201)

PS-ACSZ

 

B 012(h)

PS-ACO Ci! - Q

 

O
h
N
0

d
O

73
ACC Oxidase Activity (1)
(nl C,H, h ‘ 9" FW)

 

 

ACC Synthase Activity (e)
(pmol ACC h ‘ 9" FW)

 

 

0

 

Duration of IAA Treatment (h)

Figure 3.6. Comparison of rates of accumulation of ACC synthase (0) and ACC
oxidase (V) transcript and enzyme activity after auxin treatment.

74

Efi’ect of ethylene pretreatment on IAA-induced ethylene biosynthesis

ACC oxidase enzyme activity increases following IAA treatment. It is possible
that the change in ACC oxidase contributes to the overall increase in the rate of ethylene
production in the tissue. If so, ACC synthase cannot be considered the sole, rate-limiting
enzyme in ethylene biosynthesis. To address this possibility, seedlings were pretreated
for 4 h with 40 ILL/L ethylene to raise the levels of ACC oxidase before treating the
seedlings with IAA. If the level of ACC oxidase is initially limiting, the rate of ethylene
production should increase more rapidly in mdlings pretreated with ethylene before IAA
treatment than in swdlings treated with IAA alone. Figure 3.7 shows that ethylene
pretreatment results in increased ethylene production. As a control, ACC synthase
activity was measured to determine if the change in ethylene production could be
attributed solely to the increased levels of ACC oxidase. Unexpectedly, the ethylene
pretreatment before the application of IAA resulted in elevated ACC synthase activity and

transcript abundance (Figure 3.8).

75

 

3.2
e
2.4 - / \ 4" °2H4
+
IAA
1.6 -

Rate of Ethylene Production
(nl h" g“FW)

 

 

 

 

Figure 3.7. Effect of ethylene pretreatment on rate of auxin-induced ethylene production.
Intact seedlings were placed in desicators with (O) or without (0) 40 uL/L ethylene for
4 h. The seedlings were then treated with auxin as described in Figure 3.3. At the times
indicated, l-cm sections were isolated from the first intemode and used for measuring
ethylene production. The experiment was performed three times with similar results.

76

- + + 4h 02H,
+ - + 2hIAA

ACS Activity 2.2 0.6 13.0

 

PS-ACSZ

 

Figure 3.8. Effect of ethylene pretreatment on auxin-induced ACC synthase activity and
transcript abundance. A portion of the sections used in Figure 3.7 were used for ACC
synthase activity assays and RNA isolation. RNA blot analysis was done as described
in Figure 6.6, except that 30 pg of total RNA was loaded in each lane. The experiment
was performed twice with similar results.

77

Discussion

Most of the studies on the control of ethylene production have focused on the
regulation of ACC synthase, primarily because it has been considered the rate-limiting
enzyme of the pathway. Recent evidence indicates that ethylene-inducing stimuli also
cause an increase in ACC oxidase activity (Kende, 1993). Therefore, the effect of IAA
and ethylene on ACC oxidase in the first intemodes of etiolated peas was examined.

Before Ververidis and John (1991) determined how to recover in vitro ACC
oxidase enzyme activity, the only recourse was to determine ACC oxidase activity in vivo
by incubating tissue with high concentrations of ACC for 1 to 3 h and measuring the
amount of ethylene produced. This in vivo method has two disadvantages. First,
isolating stem sections induces, over time, a wound response which has been shown to
increase ACC oxidase activity in winter squash mesocarp (Hyodo et al., 1993) and
etiolated mung bean hypocotyls Kim and Yang (1994). Second, ACC oxidase activity
continues to increase during the incubation period of the in vivo assay. Because the
enzyme activity is changing, any value measured is an average between two time points
and cannot be attributed to any one time point on a curve. This complicates comparisons
between activity levels and transcript abundance because the RNA is isolated at specific
time points.

To exclude the effects of wounding, it was important to treat intact seedlings with
the auxin solutions. Previously, IAA-induction studies involved incubating tissue sections

on IAA solutions that often contained a cytokinin to maximize changes in the normally

78
low ACC synthase activity (Kim et al., 1992; Yoshi and Imaseki, 1982). It is now

apparent, however, that this type of treatment can cause both wound-induced and IAA-
induced transcription of different ACC synthase genes, with unknown effects resulting
from the benzyladenine treatment. The multiple gene products could contribute to
changes in ACC synthase activity and ethylene production. The obvious complexity of
these types of experiments complicates the interpretation of results and demonstrates the
necessity of using intact mdlings with gentle application of test solutions to specifically
study the effects of auxin on ethylene biosynthesis.

A ten-fold difference between the ethylene-induced ACC oxidase transcript
abundance and enzyme activity was observed. The difference is most likely a result of
the underestimation of the enzyme activity. As reported previously by Dong et al.
(1992), the addition of CO, to the reaction mixture greatly stimulates ACC oxidase
activity (data not shown). However, since the level of CO, within the tissue was not
known, and since ACC oxidase activity could be assayed without the addition of C02,
all experiments were performed at ambient CO, concentrations. Moreover, Smith et al.
(1994) have shown that the enzyme undergoes oxidative damage in the presence of ACC,
Fe“, and ascorbate, with a half-life of less than twenty minutes. Thus, the enzyme
activity would have been substantially lowered by the end of the 1 h incubation period.
As discussed by Smith et al. (1994), questions still remain about the conditions that
should be used for in vitro ACC oxidase enzyme assays.

From the data presented in this work, a model for the sequential induction of the

ethylene biosynthetic enzymes after IAA treatment is proposed. IAA causes an increase

79

in ACC synthase transcript abundance leading to an increase in ACC synthase activity.
The newly formed ACC is converted to ethylene by a low, constitutive level of ACC
oxidase. The ethylene produced then causes an increase in the levels of ACC oxidase
transcript and activity via a positive fwdback loop (Figure 3.9).

The question remains as to whether the increase in ACC oxidase activity
contributes to the higher level of ethylene production. The rate of ethylene production
increases steeply between 2 and 4 h after IAA treatment (Figure 3.3), which is approxi-
mately the same time when ACC oxidase activity begins to increase. While this result
indicates that the increase in ACC oxidase activity and the increase in ethylene
production rate are related, the evidence is correlative.

While attempting to determine if ACC oxidase contibutes to changes in the rate
of ethylene production, an unexpected result was observed. If the tissue was pretreated
with ethylene to increase the ACC oxidase activity, the ACC synthase transcript
abundance and activity was super-induced by IAA treatment. Because ethylene treatment
alone does not affect ACC synthase activity or transcript level (Figure 3.8), the ethylene
pretreatment must alter the response to the IAA treatment. One explanation is that the
increased levels of ACC oxidase would convert more of the ACC to ethylene, possibly
relieving product inhibition on ACC synthase enzyme activity. This hypothesis,
however, would not explain the increased levels of ACC synthase transcript that is also

observed.

Methionine

SAM Synthetase l

SAM
ACC Synthase 1

ACC
ACC Oxidase 1+ ‘ .................

 

Ethylene ................. ,

Figure 3.9. Positive feedback of ethylene on ACC oxidase after auxin treatment.

81

Another possibility is that the increase in ACC synthase activity is a result of the
step-down in the ethylene concentration from 40 uL/L ethylene during the pretreatment
to ambient concentrations (0.001 uL/L) during the IAA treatment, suggesting an adaptive
response in ethylene perception. This possibility is intriguing because ETR, the ethylene
receptor, resembles proteins of the prokaryotic two-component system of signal
transduction (Chang et al., 1993). Some of these bacterial regulators, such as those in
the chemotaxis pathway utilizing CheA and CheY in 1E. coli, utilize dynamic protein
phosphorylation cascades in adaptive responses (Borkovich and Simon, 1990). If
ethylene responses also involve adaptation, the increase in ACC synthase activity
following the ethylene pretreatment might involve a decrease in the level of a functional
inhibitor as the tissue undergoes deadaptation. It would be interesting to investigate
whether other IAA-inducible genes are similarly affected or if the response is specific for

ACC synthase.

82

References

Abeles FB, Morgan PW, Saltveit ME (1992) Ethylene in Plant Biology. Academic
Press, New York

Botella JR, Arteca JM, Schlagnhaufer CD, Arteca RN, Phillips AT (1992)
Identification and characterization of a full-length cDN A encoding for an auxin-
induced l-aminocycopropane-l-carboxylate synthase from etiolated mung bean
hypocotyl segments and expression of its mRN A in response to indole—3-acetic
acid. Plant Mol Biol 20: 425-436 ‘

Borkovich KA, Simon MI (1990) The dynamics of protein phosphorylation in bacterial
chemotaxis. Cell 63: 1339-1348

Burg SP, Burg EA (1966) The interaction between auxin and ethylene and its role in
plant growth. Proc Natl Acad Sci USA 55: 262-269

Chang C, Kwok SF, Bleecker AB, and Meyerowitz EM (1993) Arabidopsis ethylene-
response gene E7711: Similarity of product to two-component regulators. Science
262: 539-544

Dong JG, Fernandez-Maculet J C, Yang SF (1992) Purification and charact erization
of l-aminocyclopropane-l—carboxylate oxidase from apple fruit. Proc Natl Acad
Sci USA 89: 9789-9793

Drory A, Mayak S, Woodson WR (1993) Expression of ethylene biosynthetic pathway
mRNAs is spatially regulated within carnation flower petals. J Plant Physiol 141:
663-667

Holdsworth MJ, Bird CR, Ray J, Schuch W, Grierson D (1987) Structure and
expression of an ethylene-related mRNA from tomato. Nuc Acids Res 15: 731-
739

Hyodo H, Hashimoto C, Morozumi S, Hu W, Tanaka K (1993) Characterization and
induction of the activity of l-aminocylopropane-l-carboxylate oxidase in the
wounded mesocarp tissue of Cucurbita maxima. Plant Cell Physiol 34: 667-671

Jones J F, Kende H (1979) Auxin-induced ethylene biosynthesis in subapical stem
sections of etiolated seedlings of Pisum sativum L. Planta 146: 649-656

Kende H (1993) Ethylene biosynthesis. Annu Rev Plant Physiol Plant Mol Biol 44: 283-
307

83

Kim WT, Silverstone A, Yip WK, Dong JG, Yang SF (1992) Induction of 1-
aminocyclopropane-l-carboxylate synthase mRNA by auxin in mung bean
hypocotyls and cultured apple shoots. Plant Physiol 98: 465-471

Kim WT, Yang SF (1994) Structure and expression of cDNAs encoding l-aminocyclo-
propane-l-carboxylate oxidase homologs isolated from excised mung bean
hypocotyls. Planta 194: 223-229

Lizada MCC, Yang SF (1979) A simple and sensitive assay for l-aminocyclopropane-l-
carboxyllic acid. Anal Biochem 100: 140-145

O’Neill SD, Nadeau J A, Zhang XS, Bui AQ, Halevy AH (1993) InterOrgan regulation
of ethylene biosynthetic genes by pollination. Plant Cell 5: 419-432

Peck SC, Olson DC, Kende H (1993) A cDNA sequence encoding l-aminocyclo-
propane-l-carboxylate oxidase from pea. Plant Physiol 101: 689-690

Puissant C, Houdeline L-M (1990) An improvement of the single-step method of RNA
isolation by guanidinium thiocyanate—phenol-chloroform extraction. Biotechniques
9: 148-149

Riov J, Yang SF (1982) Effects of exogenous ethylene on ethylene production in citrus
leaf tissue. Plant Physiol 70: 136-141

Saltveit Jr ME, Dilley DR (1978) Rapidly induced wound ethylene from excised
segments of etiolated Pisum sativum L., cv. Alaska. 1. Characterization of the
response. Plant Physiol 61: 447-450

Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: A laboratory manual.
Cold Spring Harbor Laboratory, Cold Spring Harbor, New York

Schierle J, Schwark A (1988) Asymmetric synthesis and concentrations of ethylene in
the hypocotyl hook of Phaseolus vulgaris. J Plant Physiol 133: 325-331

Schierle J, Rohwer F, Bopp M (1989) Distribution of ethylene synthesis along the
etiolated pea shoot and its regulation by ethylene. J Plant Physiol 134: 331-337

Schwark A, Schierle J (1992) Interaction of ethylene and auxin in the regulation of hook
growth I: The role of auxin in different growing regions of the hypocotyl hook
of Phaseolus vulgaris. J Plant Physiol 140: 562-570

Sisler EC, Yang SF (1984) Anti-ethylene effects of cis-2-butene and cyclic olefins.
Phytochemistry 23: 2765-2768

s4

Ververidis P, John P (1991) Complete recovery in .vitro of ethylene-forming enzyme
activity. Phytochemistry 30: 725-727

Yang SF, Hoffman NE (1984) Ethylene biosynthesis and its regulation in higher plants.
Annu Rev Plant Physiol 35: 155-189

Yoshi H, Imaseki H (1982) Regulation of auxin-induced ethylene biosynthesis.
Repression of inductive formation of 1-aminocyclopropane-1-carboxylate synthase
by ethylene. Plant & Cell Physiol 23: 639-649

CHAPTER 4

CLONING OF TWO cDNAs ENCODING
l-AMINOCYCLOPROPANE-l-CARBOXYLATE SYNTHASE
INDUCED BY INDOLE-3-ACETIC ACID IN ETIOLATED PEAS

85

86
Abstract

Two cDNA clones of IAA-induced ACC synthase, PS-ACSl and PS-ACS2, were isolated
from a cDNA library made from the apical hooks of etiolated pea seedlings treated for
4 h with 10“ M IAA. The transcript level of PS-ACSl increased 75-fold in the third
intemode of IAA-treated etiolated pea stems. Simultaneous treatment with ethylene
inhibited this increase by almost 90%. Similarly, the transcript of PS—ACS2 increased
15-fold after IAA treatment, and ethylene caused a 70% inhibition of transcript
accumulation. The inhibition of IAA-induced transcript accumulation by ethylene is
consistent with the 70% decrease in the ACC synthase enzyme activity. These results
indicate that ethylene inhibits its own biosynthesis by decreasing ACC synthase levels via
a negative feedback loop. It was observed that PS-ACSl hybridized to two transcripts
on RNA blot analysis, one of 1.6 kb and one of 1.9 kb. Results of genomic DNA blot
analysis and RNA blot analysis using the 3’-untranslated region as a probe indicate that
both transcripts are derived from a single gene. Sequencing the ends of eight
independent clones of PS-ACSl showed what appears to be three, unique polyadenylation
sites. Moreover, oligonucleotide-directed RNase H mapping of the PS-ACSl transcripts
indicates that the larger transcript contains an additional sequence in the 5’-untranslated

region.

87
Introduction

ACC synthase catalyzes the first committed step of ethylene biosynthesis, the
formation of ACC from AdoMet. The enzyme was first identified in homogenates of
ripening tomato pericarp tissue by Boller et al. (1979) and Yu et a1. (1979). Its activity
was shown to be enhanced by factors which stimulate ethylene production, such as
wounding, ripening, and auxin (Yang and Hoffman, 1984). Soon after Sato and
Theologis (1989) isolated the first cDNA clone for ACC synthase by screening an
expression library with antibodies, ACC synthase cDNAs were isolated from a number
of different plants (for a review, see Kende, 1993). It was shown that ACC synthase
was encoded by a multigene family whose members were differentially regulated,
primarily at the transcriptional level, by the various factors which stimulate ethylene
production (Kende, 1993). While the amino acid sequence identities vary from 48-97% ,
seven regions are highly conserved among all ACC synthases (Kende, 1993). These
regions provide target sequences for designing oligonucleotide primers to be used in
PCR, facilitating the cloning of new ACC synthase cDNAs.

In etiolated pea seedlings, an asymmetric distribution of ethylene biosynthesis may
be involved in the asymmetry of growth that leads to the formation of the apical hook
(Schierle and Schwark, 1988). Because ethylene production in vegetative tissues is
thought to be regulated by the level of free IAA (Yang and Hoffman, 1984), the
asymmetry in ethylene production might result from a gradient of auxin concentration

between the outer and inner portions of the apical hook. To understand how these two

88

hormones may interact in this response, the effects of IAA on the enzymes of ethylene
biosynthesis are being studied. Rather than attempting to catalogue the factors
influencing the expression of a particular gene in all parts of the plant, the present work
concentrated on studying the effects of auxin only in etiolated pea seedlings, a well-
characterized system for studying ethylene production (Abeles et al. , 1992). The rapid
response time (changes occur within 0.5 h and are cOmpleted by about 8 h) negates
concerns about developmental changes in the tissue that might arise if the response
continued over several days. The use of etiolated tissue eliminates the complications
light-induced changes in development and sensitivity to various growth regulators.
Finally, as discussed in Chapter 3, the application method of gently spraying the auxin

onto the seedlings reduces the contribution of wound-induced changes.

89
Materials and Methods

Plant material and hormone treatments
Growth of pea maings and treatment with hormones were as described in

Chapter 3. In all experiments, sections were isolated from the third Orighest) intemode.

Cloning and sequencing of cDNA

PCR-based amplification was carried out using the primers described in Chapter
3. A cDNA library (Stratagene, La Jolla, CA) made from poly(A)+ RNA from apical
hooks of etiolated peas treated for 4 h with 10" M IAA and cDN A obtained via reverse
transcription of RNA from the third intemode of etiolated peas treated for 2 h with 10“
M IAA were used as templates. Reaction conditions and procedures were as described
in the Materials and Methods section of Chapter 3. The two PCR products obtained,
PACCl and PACC2, were used to screen the unamplified pea cDNA library. Inserts of
the longest full-length cDNAs hybridizing to each PCR product, PS-ACSl and PS-ACS2

were sequenced completely on both strands.

Cross-hybridization of PS-A CS1 and PS-A CS2

To determine the degree to which the two ACC synthase probes would cross-
hybridize, control experiments using sense-strand RNA from each of the clones were
performed. For PS-ACSl, the plasmid was digested with Eco R1 to linearize the vector,

and the sense strand was produced by in vitro transcription using T3 RNA polymerase.

90
For PS-ACS2, Barn HI was used to linearize the plasmid, and T7 RNA polymerase was

used to produce the sense strand. Visualization of the sense-strand products on ethidium
bromide-stained agarose gels was used to confirm that both templates produced
approximately equivalent amounts of transcript. Serial dilutions of each reaction were
separated on 1.2 % formaldehyde-agarose gels, blotted, and probed with a 32P-labeled
anti-sense strand of either PS-ACSl or PS-ACS2. The signals were quantified with a

phosphorimager (Molecular Dynamics, Sunnyvale, CA).

ACC synthase assays
Enzyme assays were performed as described in Chapter 3 except that the

concentration step with PEG was not needed.

RNA blot analysis

RNA isolation and RNA blot analysis was done as described in Chapter 3.
Unless otherwise noted, probes were the full anti-sense RNA strand of either PS-ACSl
or PS-ACS2 labeled with 32P-UTP. For the 3’-untranslated region (UTR) probe of PS-
ACSl, a 340-bp See I fragment of PS-ACSl containing only the 3’-UTR was subcloned

into pBSK‘ which was used to produced the anti-sense RNA probe.

Genomic DNA blot analysis
Genomic DNA was isolated from pea leaves as described by Sambrook et al.

(1989). Ten ug of genomic DNA was digested with Bam HI, Eco R1, or Hind HI,

91
separated on a 0.7% agarose gel, and blotted to Hybond-N membrane (Amersham).

Prehybridization and hybridization were performed at 65°C in 5X SSC, 5X Denhardt’s
solution, and 0.5 % SDS. The probe was generated by random prime labeling with 3”P-
aCTP using the full insert of pPS-ACSl as template. Final wash conditions were 0.2X

SSC, 0.5% SDS at 65°C.

Oligonucleotide-directed RNase H mapping of PS-A CSI transcript

Twenty pg of total RNA was mixed with either 2 [lg oligo d(T), 250 ng of
oligonucleotides designated SEQB (5’-CAAGA'ITCGTCCTA'ITI’CCT-3’) and SEQC
(5’-GTGGATI'TGATGGAT1'I‘GTG-3’), or water. The total volume was brought to 25
pL with water. The mixture was heated to 65 °C for 5 min before slow cooling to room
temperature. Ten uL 5X RNase buffer (1X = 20 mM KCl and 4 mM Tris-HCl, pH
8.0), 6.5 11L sterile DEPC-treated H20, 5 uL 100 mM MgC12, 2.5 uL 20 mM DTT, and
l uL 1.0 unit/ILL RNase H were added to a total of 50 ILL. This mixture was incubated
at 37 °C for 45 min. The entire reaction was then precipitated with 5 [L 3 M NaOAc,
pH 4.8, and 50 uL isopropanol at -20°C for l h. The pellet was resuspended in RNA
loading buffer (Sambrook et al., 1989) and used for RNA blot analysis as described

above.

Results

Isolation of cDNAs for two auxin-induced ACC synthases

To isolate cDN A clones for ACC synthase transcripts expressed in etiolated
seedlings after auxin treatment, PCR was used to amplify fragments containing the active
site region. Degenerate oligonucleotide primers based on conserved regions surrounding
the active site (TNPSNPLGT and MSSFGLVS; Kende, 1993) were used in PCR with
two templates, a cDNA library from etiolated pea apical hooks treated for 4 h with 0.1
mM IAA and cDNA obtained by reverse transcription of mRN A from first intemodes
of etiolated seedlings treated for 2 h with 0.1 mM IAA. By this method, two unique
PCR fragments, PACCl and PACC2, were isolated. Further attempts to isolate cDNAs
for other ACC synthases yielded only these two fragments, indicating that PACCl and
PACC2 represented the two predominant transcripts expressed in etiolated seedlings
following IAA treatment.

These fragments were used to screen 600,000 plaques from the unamplified,
cDNA library from IAA-treated apical hooks. Thirty-two independent clones which
hybridized with PACCl were isolated. The sequence of PS-ACSl, the longest cDNA
clone hybridizing with PACCl, is given in Figure 4.1. It contains a predicted open
reading frame of 1443 bp (481 amino acids), a 375-bp 3’-untranslated region (UTR), and
a 245-bp 5’-UTR. Of the eight cDNAs sequenced, the three longest clones shared 133

bp of identical 5’-UTR.

93

Legend for Figure 4.1. The shaded region shows the variable sequence. The alternative
sequences (U102 and U11) for the 5’-end are given at the top of the figure.

0102

011

94

(GCCATGTTCATGTTTCAATTCCAAACCAAACCAAGACATGGACCTAACATTCATCATACACGATT
ATTCAAAACCACACTCAAATTCAAAACTATAAACTTT)
(TATAAAGCATAGTTATATTTATAGTAATAATACCTCAAACTTT)

1 i131}:...:ii‘.;:izl.;ii2._'éi. Iiinfijff1.ffif-f..fl..f.51 .3. if Lfi'..ff.._[ ” " '
67 "T

133
199

265
331
397
463
529
595
661
727
793
859
925
991
1057
1123
1189
1255
1321
1387
1453
1519
1585
1651
1717
1783
1849
1915

1981
2047

 

CATACATTGCTTTTCATTGTTTCATTTCTTACTCCTACAAAAGAAAATGAAGTTGCTATCTACAAA
M K L L S T K
AGCCACATGCAACTCTCATGGCCAAGATTCGTCCTATTTCCTAGGATGGCAAGAATATGAAAAAGA
A T C N S H G Q D S S Y F L G W Q E Y E K E
AAACCCTTATTATCATGTTCAAAATCCCAAAGGAATTATTCAGATGGGTCTCGCCGAAAATCAGCT

N P Y Y H V Q N P K G I I Q M G L A E N Q L
GTCTTTTGATCTATTAGAATCATGGCTTGCTAAGAATCAAGATGTAGGAGGATTCAAACGTGATGG
S F D L L E S W L A K N Q' D V G G F K R D G
AAAATCAATATTTAGAGAACTTGCTCTCTTCCAAGACTACCATGGTCTACCATCTTTCAAGAAAGC
K S I F R E L A L F Q D Y H G L P S F K K A
ATTGGTTGATTTCATGGCTGAGATTAGAGGAAACAAAGTCACTTTTGATCCAAACCACATTGTTCT
L V D F M A E I R G N K V T F D P N H I V L
AACAGCTGGTGCCACTTCTGCTAATGAGACACTCATGTTTTGTCTTGCTGAAAAAGGAGAAGCCTT
T A G A T S A N E T L M F C L A E K G E A F
TCTTCTTCCCACTCCCTATTATCCAGGATTTGATAGAGACTTGAAGTGGAGAACTGGGGTGGAGAT
L L P T P Y Y P G F D R D L K W R T G V E I
AGTTCCAATACAATGCACAAGTTCAAACAATTTCCAAATGACTGAATCTGCTTTGCAACAAGCTCA
V P I Q C T S S N N F Q M T E S A L Q Q A H
TGAAGATGCAAAGAAGAAGAACCTAAAAGTCAAAGGTGTCTTAGTCACAAATCCATCAAATCCACT
E D A K K K N L K V K G V L V T N P S N P L
AGGCACTACAATGTCAAAGAATGAATTAAACCTTCTCATTGACTTCATCAAAGACAAAAACATGCA
G T T M S K N E L N L L I D F I K D K N M H
TTTAATAAGCGACGAGATTTACTCCGGTACAGTTTTCACTTCTCCAAACTTCGTCAGCGTCATGGA
L I S D E I Y S G T V F T S P N F V S V M E
AATCCTTAACGAAAGAACCGATAAGGATTTCCTCGACGCTAACGTCTCAGAAAGAGTTCACATCGT
I L N E R T D K D F L D A N V S E R V H I V
CTATAGTCTCTCCAAAGATCTCGGTCTACCAGGCTTCCGTGTCGGCGCTATCTATTCCGACAACGA
Y S L S K D L G L P G F R V G A I Y S D N E
AACCGTCGTTGCAGCCGCGACGAAAATGTCTAGCTTTGGTTTGGTTTCATCACAAACTCAATACCT
T V V A A A T K M S S F G L V S S Q T Q Y L
TCTCTCAGCTATGCTAGGTGACAAAAAATTCACAAGAAACTACTTATCCGAGAATCAAAAAAGACT
L S A M L G D K K F T R N Y L S E N Q K R L
CAAAAAACGACAGAAAATGCTTGTTAACGGATTGCAAAAAGCCGGTATTAGCTGTCTGAAAACAAA
K K R Q K M L V N G L Q K A G I S C L K T N
CAACGCTGGTTTGTTTTGTTGGGTTGATATGAGAAATCTTCTAACATCAGACACATTCGAAGCTGA
N A G L F C W V D M R N L L T S D T F E A E
AATGGATTTATGGAAGAAGATATTATACGAAGTTGGGTTGAACATTTCACCAGGTTCATCATGTCA
M D L W K K I L Y E V G L N I S P G S S C H
TTGCACTGAACCAGGTTGGTTTCGTGTTTGTTTTGCTAACATGTCGGAAGATACATTAAACCTAGC
C T E P G W F R V C F A N M S E D T L N L A
TATGAAAAGGTTAAAGGACTTTGTCTCAAACTCCAACGGTGAAGAGGGTAGTAATAGTGATAACAA
M K R L K D F V S N S N G E E G S N S D N K
GAGAACTAGAAGTTCCCAATCTTCTAGAAGTTTTACAAGAAAATCGATTTCGAATTGGGTTTTTAG
R T R S S Q S S R S F T R K S I S N W V F R
GTTATCTTCTCGTGATCATCATGAACAAGAAGAACGATAACCTGGTTAACTATGGTGTGAATGTGA

L S S R D H H E Q E E R *
AGTACTACGCATGCATGCACAATAGTATCTATTACTATTAACTTTTTATTTTTATTGTTTTTTTAC
ACTCCTTGGCCAAATATTTTTTGGGGGGATTTTAGGATCTTACTTTTGGAAAAATATATATTGTAA
TGGTAAATTGTAGAGGGAAGGAAATTAAGAAAAACTCACTCTGTCCATTTGGAATTGAAGTGTGAG
GTGTGTAGGATTATGACAAACTTTAAAGTGTGGTTGAAAAATGTTGTTGGTGTGAATGTGATGGAT
GGAAAGTTTGAGGTATTATTACTATAAATATAACTATTGCTTTATATTTCCGTCCTCATTTTTTAT
AAAAAAAAAAAAAAAAAA

Figure 4.1. Nucleotide and deduced amino acid sequence of PS-ACS1.

95
The remaining 39 bp (clone ACSl-U11), 92 bp (clone ACS1-U102), and 113

bp (ACSl-14; shown as the shaded region of PS-ACSl in Figure 1) of the 5'-
UTRs were unrelated. Isolation of a genomic clone for PS-ACS1 will be
necessary to determine which of these 5’-untranslated sequences are correct.
The 3'-UTRs of the eight cDNAs sequenced differed only in length, falling into
three, distinct groups (Figure 4.2). Each of thesegroups had one cDNA which
contained a polyiA)-tail, suggesting that these groups may represent true
differences in polyadenylation sites. The sequence of PS-ACS1 predicts a
hairpin structure formed by basepairs 978 to 1035 (Figure 4.3). This structure
would contain a 26-bp stern and 7-bp loop, with a predicted 116 of -38 kcal
(Sasker, 1977).

Five independent clones hybridizing with PACC2 were isolated. The
sequence of the longest cDNA hybridizing with PACC2, PS-ACSZ, is given in
Figure 4.4. The sequence contains a predicted Open reading frame of 1461 bp
(487 amino acids), a 137-bp 5’-UTR, and a 251-bp 3’-UTR.

Using in vitro transcribed sense-strand RNAs as controls, it was
determined that the antisense RNA strand probes for the two clones show less
than 0.01% cross-hybridization with each other (Figure 5). Thus, the
expression patterns of the two genes coding for PS-ACS1 and PS-ACSZ can

be distinguished by RNA blot analysis.

Group I

ACSl-UZl
ACSl-lS
ACSl-UIOZ
ACSl-B

Group II

ACSl-U72
ACSl-A122
ACSl-Ull

Group III

ACSl-14

1950 1960 1970
TTGAAAA
TTGAAAAATGTTGTT

TTGAAAAATGTTGTTGGTGTG(A)w
TTGAAAAATGTTGTTGGTGTGAATGTGAT

1970 1980 1990 2000 2010 2020
GAATGTGATGGATGGAAAGTTTGAGGTATTATTACTATAAATATAACTATGC
GAATGTGATGGATGGAAAGTTTGAGGTATTATTACTATAAATATAACTATGCTT
GAATGTGATGGATGGAAAGTTTGAGGTATTATTACTATAAATATAACTATGCTTT(A)fl

2020 2030 2040
GCTTTATATTTCCGTCCTCATTTTTTAT(A)w

Figure 4.2. 3’ ends of eight PS-ACS1 clones. The sequences of all the shorter
clones in Groups I and II were identical to the internal sequence of the longest
clone PS-ACS1-14 (Figure 4.1) from which the numbering of base pairs was
taken. A poly(A) tail was present in each of the three groups.

QO>>COOO>OOCOOCCC>QQ>>C>00

C00c0>000c0>c00>>>c00cc>>00

”C

U

PS-ACS1

O
C

Figure 4.3. Predicted hairpin structure formed by base pairs 978 to 1035 of PS-
ACSl.

1

61
121
181
241
301
361
421
481
541
601
661
721
781
841
901
961
1021
1081
1241
1301
1361
1421
1481
1541
1601
1661
1721
1781

1841
1901

98

CTTCAAGCTATAAATAACCACCCATACTTCATTCCTAGATATCATTGCAATCAATCTCTC
AACTACAAACACAACAATTTTACTTTCTTTGCTAATATCACACTACATTAATCATTTTTA
TACTACTATAATTAACAATGGGAGTGATGAACTTGGATCAACCACAATTGTTGTCTAAGA
M G V M N L D Q P Q L L S K I
TAGCCATGGGTGATGGCCATGGTGAAGCATCATCTTACTTTGATGGATGGAAAGCCTATG
A M G D G H G E A S S Y F D G W K A Y D
ACAAAGATCCTTTCCATCCATCCAAAAATCCCCACGGAGTCATCCAAATGGGTCTTGCAG
K D P F H P S K N P H G V I Q M G L A E
AGAATCAGCTTACTGCTGATATGGTTCAAAATTGGATTATGAGTAATCCAGAAGCTTCGA
N Q L T A D M V Q N W I M S N P E A S I
TTTGTACGCTAGAAGGAGTTCATAATTTCAAACAGATGGCGAATTTTCAGGATTATCATG
C T L E G V H N F K Q M A N F Q D Y H G
GTTTACCAGAGTTCAGAAATGCTGTGGCGAAATTCATGTCTAGAACCAGAGGAAACAGAG
L P E F R N A V A K F M S R T R G N R V
TGACGTTCGATCCCGAACGGATCGTAATGAGCGGCGGAGCCACCGGAGCTCATGAGGCTA
T F D P E R I V M S G G A T G A H E A T
CGGCGTTTTGTTTGGCGGATCGTGGTGAAGCTCTTTTGGTGCCTACTCCTTATTATCCAG
A F C L A D R G E A L L V P T P Y Y P G
GTTTTGATAGAGATTTGAGGTGGAGAACAGGAGTTAAACTTGTACCAGTTATCTGTGAAA
F D R D L R W R T G V K L V P V I C E S
GCTCAAACAATTTCAAATTAACCAAACAAGCCTTAGAAGAAGCTTATGAAAAAGCCAAAG
S N N F K L T K Q A L E E A Y E K A K E
AAGATAACATCAGATTCAAAGGTTTACTCATCACAAATCCTTCAAATCCTTTAGGCACTG
D N I R F K G L L I T N P S N P L G T V
TTATGGACAGAAACACACTAAGAACTGTCATCACTTTCATCAACGAAAAGCGTATCCATC
M D R N T L R T V I T F I N E K R I H L
TAATAAGCGACGAAATCTACGCTGCAACGGTTTTTAGCCACCCAAGTTTCATAAGCATAG
I S D E I Y A A T V F S H P S F I S I A
CGGAGATAATCGAACATGACACAGACATTGAATGCGACCGTAACCTCGTTCACATAGTTT
E I I E H D T D I E C D R N L V H I V Y
ATAGTCTTTCAAAAGACATGGGATTCCCGGGGTTTAGAGTTGGTATAATATACTCATACA
S L S K D M G F P G F R V G I I Y S Y N
ACGACACCGTTGTTGATTGCACGCGCAAAATGTCGAGTTTCGGACTAGTTTCAACACAGA
D T V V D C T R K M S S F G L V S T Q T
CACAGTATTTGATCGCGAAAATGCTATCCGATGATGACTTCGTCGAGAAATTTCTTCCCG
Q Y L I A K M L S D D D F V E K F L P E
AGAGTGCGAAGAGGTTAGCACAAAGGTACAGAGTTTTCACGGGCGGATTAATCAAAGTCG
S A K R L A Q R Y R V F T G G L I K V G
GAATCAAGTGTTTGCAAAGTAACGGTGGACTTTTCGTGTGGATGGATTTAAGAGGACTTC
I K C L Q S N G G L F V W M D L R G L L
TTAAGAATGCAACATTCGAATCAGAAATCGAACTATGGAGAGTGATTATTCATGAAGTTA
K N A T F E S E I E L W R V I I H E V K
AGATCAATGTTTCACCTGGTGTTTCATTTCATTGCTCTGAACCAGGTTGGTTTAGAGTGT
I N V S P G V S F H C S E P G W F R V C
GTTATGCTAACATGGATGATAGAGATGTGCAAATTGCATTACAAAGGATTAGATCATTTG
Y A N M D D R D V Q I A L Q R I R S F V
TGACTCAGAATAACAAAGAGGCTATGGGTTCTGATAAGAACTCTAAACCTTACTGGCATA
T Q N N K E A M G S D K N S K P Y W H S
GTAATTTGAGGTTGAGCCTTAAACCAAGAAGGTTTGATGATATTATGATGTCACCTCATT
N L R L S L K P R R F D D I M M S P H S
CTCCAATTCCTCAATCACCTCTTGTGAAAGCTACTACTTGAATTGATTACATGGTTTTTA
P I P Q S P L V K A T T *
GTATCATATAGATTATGAAGAAATAACTGATAGAAGATTCTTTGGTTTGTTGATTTATTA
TTTTAGGAGCCTAGATAATAGGTCCTTTAAATGTTGGGGCATTTTTTTTCTTTTTTCACT
TGTTGAATCACATTGTGGTGAAAGAAGTTTATAGAATTTGTAAGGCTATTGTTTGTAATT
TGTTTGAAGGGTTCAGAAAATATCTGTAATCTGTTTTATGAAATTGTTA

Figure 4.4. Nucleotide and deduced amino acid sequence of PS-ACSZ.

Ps-Acsz PS-ACSl

 

 

161162163104 162115310" (11L)

..-

PS-ACS1 PS-ACSZ

 

 

161162163104 162163104 (11L)

0" .

Figure 4.5. Cross-hybridization of PS-ACS1 and PS-ACSZ. The cDNA clones
were used to produce sense strand RNA by in vitro transcription. The
transcription reaction yielded approximately equal amounts of the sense- strand
product as determined by visualization on an ethidium bromide stained agarose
gel. Serial dilutions of the sense-strand transcription reactions were separated
on a 1.2% agarose-formaldehyde gel, blotted, and probed with 32P-labeled
antisense RNA prepared from either (A) pPS-ACS1 or (B) pPS-ACSZ.

100

Effect of IAA and 02H, on ACC synthase expression and activity.

Extractable ACC synthase activity (Figure 4.6) and transcript levels of
both PS-ACS1 and PS-ACSZ (Figure 4.7A) increased in the third intemode of
6 to 7 d-old intact etiolated seedlings after treatment with 0.1 mM lAA. While
the transcript level of PS-ACS1 reached a maximum at 4 h and then declined
to almost undetectable levels by 6 h, the level of PS-ACSZ remained elevated
over the course of the experiment (Figure 4.7A). Transcript levels of PS-ACSl
increased 75-fold after IAA-treatment (Figure 7A) but increased only 9-fold
when the IAA-treated seedlings were treated simultaneously with ethylene
(Figure 4.73). Similarly, PS-ACSZ transcript increased 15-fold after lAA
treatment (Figure 4.7A) but only 5-fold during simultaneous ethylene treatment
(Figure 4.78). The repression of ACC synthase transcript accumulation was
consistent with the reduced levels of ACC synthase activity measured in the
IAA-treated tissue co-treated with ethylene (Figure 4.6). Treatment with a
water control of the same pH did not cause a detectable increase in ACC
synthase activity (Figure 4.6) or transcript levels of PS-ACSl (Figure 4.7C).
Levels of PS-ACSZ transcript did increase after the water treatment (Figure
4.7C), but not to the same extent as with IAA-treatment (Figure 4.7A). It has
been proposed that VR-ACSl , an ACC synthase clone from mung been with
81% amino acid identity with PS-ACSZ, is induced by touch (Botella et al.,
1995), which may explain why the level PS-ACSZ transcript increased

following the water treatment.

101

 

 

 

 

 

 

135-

>‘A

E5120-

137105-

0

(Fm ea-

33"; 75-

.50 60-

5.2

m_ 45-

O 30.

SE

(315" H20
0 V 7" l
o 2 4 6

Duration of Treatment (h)

Figure 4.6. Effect of auxin and ethylene on ACC synthase activity. At 0 h,
intact seedlings were sprayed with solutions containing 100 ,uM IAA (O, O) or
no IAA (v). The IAA-treated seedlings were placed in desiccators with (O) or
without (0) 40 pL/L ethylene. At the times indicated, 1-cm sections were
isolated from the third (highest) intemode and assayed for ACC synthase
enzyme activity. The experiment was performed twice with similar results.

102

A 0246(h)

PS-ACS1 n
PS-ACSZ . 4”

 

 

 

 

 

 

 

 

 

B o 2 4 6 (h)
PS-ACS1 ee ....
"" .
Ps-Acsz . .-
C o 2 4 6 (h)
PS-ACS1 '

 

PS-ACSZ . . .

 

 

Figure 4.7. Effect of auxin and ethylene of ACC synthase transcript levels.
Seedlings were treated with (A) 100 ,uM lAA, (B) 100 ,uM IAA + 40 pL/L
ethylene, or (C) water. Treatments were as described in Figure 4.6. RNA blot
analysis was performed as described in Figure 4.5.

103

PS-A CSI hybridizes to two transcripts.

As seen in Figure 4.7, the probe for PS-ACS1 detected two distinct
transcripts of 1.6 and 1.9 kb on RNA blots. A time course of the changes after
IAA treatment show that the 1.6-kb transcript accumulated first (Figure 4.8A).
Moreover, the two transcripts accumulated with different patterns after IAA
treatment (Figure 4.88). These results indicate that the smaller transcript is not
a degradation product of the larger.

Because of the predicted hairpin structure in PS-ACSl, the secondary
structure could lead to the appearance of two bands. In the cross-hybridization
experiment (Figure 4.5), however, the sense-strand RNA product of an in vitro
transcription reaction from pPS-ACS1 produced only a single band. Because
the in vitro transcription product had approximately the same size as the larger
transcript (data not shown), the appearance of two transcripts is probably not
an artifact of secondary structure formation.

To determine if the two transcripts are encoded by one gene or two
highly similar genes, the probe used in the RNA blot analysis was used on a
genomic DNA blot. Under stringent conditions, the full-length PS-ACSl probe
hybridized with a single band on a genomic DNA blot using three different
restriction enzymes (Figure 4.9). In addition, a 300-bp probe containing only
the 3'-UTR of the PS-ACSl cDNA hybridized with both bands on an RNA blot
(Figure 4.10). These results indicate that both transcripts are encoded by a

single gene but do not exclude a tightly linked gene duplication.

104

Legend for Figure 4.8. Seedlings were treated with 100 uM IAA as described
in Figure 4.6. (A) RNA blot analysis was performed as described in Figure 4.5.
(B) Quantitation of abundance of 1.6 kb transcript (O) and 1.9 kb transcript
(O) was done on a densitometer. The experiment was performed four times
with similar results.

 

105

A
o 0.512 4 6 8 (h)

...5; , , 41.9
W - ‘ 1.6

6000

 

5000
4000
3000

2000

RNA Abundance
(Relative Units)

1000

 

 

 

 

0 2 4 6 8
Duration of Treatment (h)

Figure 4.8. Expression pattern of two transcripts hybridizing to PS-ACS1.

106

co 8 .E
a: In I kb
it“s: V (:' ' a -. .9
i ;i} T
l gr;
““2. g 4 4.5
v.7 =9?
1 Qt -.;
:71 < 2.8
a
:21 ,_ 33‘?
7'; - ‘
* 4 1 .7
:5 3‘ , 4 1.2

Figure 4.9. Genomic DNA blot probed with PS-ACS1. Ten pg of pea genomic
DNA was digested with either Bam HI, Eco RI, or Hind III, and separated on a
0.7% agarose gel. The entire insert of pPS-ACS1 was random prime labelled

with 32P-aCTP and used as a probe. Final wash conditions were 0.2x SSC,
0.5% SDS at 65°C.

107

 

Figure 4.10. RNA blot analysis using 3’-UTR of PS-ACSI. RNA was isolated
from the third intemode of seedlings treated with 100 pM IAA for 0.5 h (lane
1) or 2 h (lane 2). The RNA was separated on a 1.2% formaldehyde agarose
gel, blotted, and probed with a 300-bp 32P-labeled antisense RNA strand from

only the 3’-UTR of PS-ACSI. The experiment was performed twice with
similar results.

108

If the two transcripts are produced from the same gene, the four most
plausable explanations for the size difference are (1 ) a difference in poly(A)-tail
length, (2) different polyadenylation sites, (3) different transcription start sites,
and (4) alternative splice sites.

RNase H mapping was used to remove the poly(A)-tail from the
transcripts. RNase H will specifically degrade RNA in a RNA-DNA hybrid.
Oligonucleotides can be used to specifically cleave RNA at the complementary
region, thus producing a shorter transcript that can be detected by RNA blot
analysis (Figure 4.1 1). Dligo d(T)15 will randomly anneal to most of the poly(A)-
tail, effectively removing the majority of the sequence. With the poly(A)-tail
removed (Figure 4.12), the transcripts shifted in size very slightly but remained
distinct, indicating that pon(A)-Iength is not responsible for the two transcripts.
A large shift would not be expected because removal of 40-65 hp, the average
steady-state poly(A)-tail length (Jacobson, 1987), would be a minor change in
1.6 to 1.9-bp transcripts. As was shown in Figure 4.2, cDNA clones for PS-
ACSi containing different polyadenylation sites were isolated. The difference
in length among the three groupings was only 100-bp, which does not appear
to be enough to account for the difference in size between the two transcripts
(about 300-bp). Different polyadenylation sites, however, may explain the
appearance of the larger transcript as a broad band on RNA blots (Figures 4.7

and 4.8).

109

RNase H Mapping

mRNA AAAAAAAA

 

OligO <——

* Anneal

w

l RNase H

 

AAAAAAAA

 

I RNA Gel/Blot

 

No
oligo oligo

 

 

 

Figure 4.11. Diagram of the procedure for RNase H mapping.

110

 

Figure 4.1 2. RNA blot analysis after removal of poly(A)-tai|. Twenty pg of total
RNA from 0.5 h (lanes 1 and 2) or 2.0 h (lanes 3 and 4) IAA-treated seedlings
were heated to 65°C and allowed to cool slowly to room temperature in the
absence of oligonucleotides (lanes 1 and 3) or in the presence of
oligonucleotide d(T)15 (lanes 2 and 4). The mix was incubated with RNase H
for 45 min at 37°C. RNA blot analysis was performed as described in Figure
4.6. The experiment was performed twice with similar results.

111

Two oligonucleotides, one 40-bp downstream of the predicted
translational start site (SEQB) and one about 600-bp downstream (SEQC), were
used in RNase H mapping of the 5' end of PS-ACS1. The primer closest to the
translational start site removed a portion of the larger band (Figure 4.13, lane
6) but did not visibly affect the size of the smaller band (Figure 4.13, lane 2).
The oligonucleotide directed to a sequence towards the middle of the transcript
caused both transcripts to be truncated (Figure 4.13, lanes 3 and 7). In fact,
with this oligonucleotide, both transcripts yielded truncated products of similar
sizes, suggesting that the difference in size between these two transcripts is
upstream of this second primer. Sequence information obtained from the
isolation of a genomic clone for PS-ACS1 will be necessary to determine if the
difference in the 5' ends is the result of different transcriptional start sites,

alternative splicing, or both.

112

Legend for Figure 4.13. Twenty pg of total RNA from 0.5 h or 2.0 h IAA-
treated seedlings was heated to 65°C for 5 min and allowed to cool slowly to
room temperature in the absence of oligonucleotides (lanes 1,4,5, and 8), in the
presence of oligonucleotide SEQB (lanes 2 and 6), or in the presence of
oligonucleotide SEQC (lanes 3 and 7). The mix was incubated with RNase H
for 45 min at 37°C. RNA blot analysis was performed as described in Figure
4.6. The experiment was performed twice with similar results.

 

 

113

0.5h 2h

 

 

 

PS-ACS1 : l

<— <—
SEQB SEQC

Figure 4.13. Oligonucleotide-directed RNase H mapping of the PS-ACS1
transcript.

114

Discussion

As was found in other plants (tomato: Yip et al., 1992; mung bean:
Botella et al., 1992, Kim et al., 1992), more than one gene encoding ACC
synthase is expressed in IAA-treated etiolated pea seedlings. It has been
suggested that the different members of the ACC synthase gene family can be
placed into groups that are highly related in both amino acid sequence identity
as well as pattern of expression (Liang et al., 1992). While both PS-ACS1 and
PS-ACSZ are regulated by IAA in etiolated pea seedlings, they share only 53%
amino acid identity. PS-ACS1, however, shares 74% amino acid identity with
LE-ACS3, an IAA-induced clone from tomato (Olson et al., 1995). As shown
in Figure 4.14, 55% of the amino acids are absolutely conserved in all members
of the LE-ACS3-type group, which includes both monocots and dicots.
Similarly, PS-ACSZ more closely resembles the group associating with LE—ACSZ
with which it shares 64% amino acid identity (Figure 4.15). Within this group,
44% of the amino acids are absolutely conserved. Between the groups of LE-
ACS3 and LE-ACSZ types, only 29% of the amino acids are conserved. The
high level of conservation of amino acids within but not between these groups
indicates that, even though the enzymes are catalyzing the same reaction in
response to auxin, there may be unique forms of regulation or constraints at the
protein level that favor one form over the other. Currently, nothing is known

about post-transcriptional regulation of ACC synthase.

115

All members of the LE-ACSS group are expressed in auxin treated
vegetative tissue, except for apple which was not examined (see references in
legend of Figure 4.14); and all members have an Arg as their C-terminal
residue. Expression seems to be primarily in the vegetative tissue, as it was
either not present (Nakagawa et al., 1991) or expressed at a low level in
ripening fruit (Dong et al., 1991; Yip et al., 1992). In tomato roots (Olson et
al., 1995) and in rice stems (Zarembinski and Theologis, 1993), expression was
increased by flooding or low oxygen. Anaerobiosis, however, did not increase
expression in etiolated Arabisapsis seedlings (Liang et al., 1992). As in the
present work, multiple bands were detected on RNA blots analysis in tomato
(Olson et al., 1995), winter squash (Nakagawa et al., 1991 ), Arabidopsis (Abel
et al., 1995), and possibly in apple (Dong et al., 1991).

The appearance of multiple bands on RNA blot analysis in other plants
is intriguing. In tomato (Olson et al., 1995), the larger transcript (2.1 kb) is a
result of introns not being spliced during flooding. Unspliced transcripts,
however, are not uncommon under stress conditions (Czarnecka et al., 1988;
Winter et al., 1988) and may be unrelated to what was observed in this work.
The results of the present work indicate that a single gene PS-ACS1 has
different transcriptional start sites, alternative splicing at the 5'-end, or both.
Because the transcripts produced in some of the other plants are doublets of
similar size as in pea, the production of two transcripts may be a conserved

response.

116

Legend to Figure 4.14. The clones are from tomato (LE-ACS3; Olson et al.,
1995), pea (PS-ACS1; this work), potato (ST-ACS1; Destafano-Beltran, 1995),
winter squash (CM-ACSZ; Nakagawa et al., 1991), Arabidopsis (AT-ACS4;
1995), apple (MS-ACS1; Dong et al., 1991, modified for this work). and rice
(Zarembinski and Theologis, 1993). Residues identical to those of LE-ACSS are
shown by "*". Gaps in the sequence are shown by "-". The original sequence
for MS-ACS1 differs from the current in that the sequence terminates
prematurely, apparently a result of a frameshift caused by a single nucleotide
addition within the shaded backslash region. Without this nucleotide,the open
reading frame presented here continues the reading frame that aligns with the
other sequences.

LE-ACSB
PS-ACSl
ST-ACSl
CM-ACSZ
AT-ACS4
MS-ACSI
OS-ACSl

LE-ACSB
PS-ACSI
ST-ACSl
CM-ACSZ
AT-ACS4
MS-ACSI
OS-ACSl

LE-ACSB
PS-ACSl
ST-ACSl
CM-ACSZ
AT-ACS4
MS-ACSI
OS-ACSl

LE-ACS3
PS-ACSl
ST-ACSI
CM-ACSZ
AT-ACS4
MS-ACSI
OS-ACSI

LE-ACS3
PS-ACSl
ST-ACSl
CM-ACSZ
AT-ACS4
MS-ACSl
OS-ACSl

LE-ACS3
PS-ACSl
ST-ACSl
CM-ACSZ
AT-ACS4
MS-ACSl
OS-ACSA

LE-ACSB
PS-ACSl
ST-ACSl
CM-ACSZ
AT-ACS4
MS-ACSl
OS-ACSI

MKLLSEKATC

*****T****
*****K**M*
**M**T****

*VQ**R****
//***K**G*

AQNPDAAGFK
*K*Q*VG***
T******A**
SK*****S**
***T***C**
*K**E**A**
EK**H*L*LR

AGATSANETL

**********
**********
**********
*******t**
*********F
********A*

LEEAYLDAKK
*QQ*HE****
*****KE*ER
**Q**K**QT
*****EQ***
*****QE*E*
*DD**RR*Q*

VFN-SP-GFV
**T-**-N**

***-**-K**
**3-**-***
***-*S-E*I
a*s-**-s*1
Atggpta***

VSAATKMSSF

*A********
**********
*A********
I*********
*A********
**********

LESN-AGLFC
*KT*N*****

****-*****
****_*****
****-*****
*NG*-*****
*p**-*****

NSHGQDSSYF

**********
**********
**********
*****v****

117

LGWWEYEK-N

***Q****E*
***E****-*
***EA**N-*
***E****-*

PYDEIQNPXG
*wyfiv*****

**D*TR****

*FHHTS**N*
**DVTK**Q*

* ***Q****-* **H*VL*TN*
********** ***Q****-* *pppvs**s*

RNGE--SIFR ELALFQDYHG LPAFKNAMTK

*D*K--****
*ttN--****
*DtK——****
*D*Q--*V**
K***--***A

*E*GGA*V**

MFCLANQGDA
*****EK*E*

*G***DP***
*****EA***
*****Dp***
I****DP*E*
*****DH***

RNLKVKGVLV

K*********
***R******
***R******
LD*N***I*I
***R******
*R*R*****I

SVMEVLI---
****I*NERT

*******__-
*A****K---
****I*K---
******K--D

*AL**VA---

GLVSSQTQYL

**********
**I*******
**********
**********
********H*
**********

WVDMRHLLSS
*****N**T*

**********
********E*
*****p**R*
********R*
****S**MR*

**********
**********
**********
**********
**********
**********

FLLPTPYYPG

**********
**********
**********
**********
V*I*******
**I*******

TNPSNPLGTT

**********
********s*
**********
**********
**********
*********A

EKNYMKTRVW
D*DFLDAN*S
*N***Y*E**
*RSSEDEE**
NNQLEN*D*L
RNCDENSE**
GRDGGGAD*S

LSCMLSDKKF

**A**G****
**AL***Q**
**A*******
**SL******
**A******L

*AAL*A*RD*

NNFDAEMDLW
DT*E******

*T**G**E**
DK*ES*LE**
KT*E******
*T*E***E**
RS*AG**E**

**s§*K*LVD
*****D*LVQ
*****K*LVE

*SS****FAD
*****K**VD

*******LAR

FDRDLKWRTG

**********
**********
**********
**********
**********
**********

LNRNELELLL
MSK***N**I
*TKK**Q***
M**D**N*VF
TTQT**NI*F
MT****Y***

SP*AD**TIV

ERVHIVYSLS
**********

D*********
K*********
n*****c***
Q***v*****
D***V*****

TKKYISENQK
*RN*L*****
MtN*v*****
*IS*******
**N*LR****
**N**A**H*
*RS*VA**KR

KKIVYDVGLN
***L*E****

*****E****
*****E****
*****E*K**
*****E*H**
**V*FE****

IIQMGLAENQ

**********
**********
**********
**********
**********
**********

FMSEIRGNRV

**A*****K*
********K*
**A*****K*
****N*****
**AK****K*
****Q**YK*

AEIVPIHCSS
V*****Q*T*
******Q*T*
V*******T*
V*****QS**
V*******T*
*****V**A*

TFIDEKG-IH
D**KD*-NM*
**VST*-Q**
D**TS*G-**
D**TKNK***
S*VED*G-**
D*VAAKG-**

KDLGLPGFRI
*********v

*********v
*********v
*********v
*********v
*********v

RLKKRHAMLV

*****QK***
******E***
***Q*QK***
***N*QRK**
***Q*QKK**
*I*E**DQ**

ISPGSSCHCT
**********

**********
**********
*********E
**********
*********R

LSFDLLESWL
**********

**********
**********
*c********
*c********
*******E**

SFDSNNLVLT
T**P*HI***
*****K****
**EA**I***
**********
T**P*H****
v**ps*1***

SNGFRITESA
**N*QM****
**********
****Q**Q**
T******KL*
****Q***T*

A****V*RA*

LISDEIYSGT

**********
**********
**********
*v********
**********
*******A**

GAIYSNDEMV
*****DN*T*

*******D*I
*******D**
*******KD*
*******D**

*****ANAA*

KGLKSAGINC
N**QK***S*
G***QI**R*
S**QK*****
L**EAI**K*
S**QKS**S*
D**REI**G*

EPGWFRVCFA
**********

******A***
**********
**********
**********
**********

Figure 4.14. Amino acid sequence comparison of ACC synthase clones similar
to LE-ACS3.

118

Figure 4.14 (continued)

LE-ACS3
PS-ACSI
ST-ACSI
CM-ACS2
BT-ACS4
MS-ACSI
OS-ACSl

LE-ACSB
PS-ACSI
ST-ACSl
CM-ACSZ
AT-ACS4
MS-ACSI
OS-ACSl

NMSEDTLDLA MRRIKDFVES TAPNATNHQN QQQSN-ANS- K---KKSFSK WVFRLSFNDR

*******N**
*******NI*
****s**x**
**IDE**K**
*LP*R*****
***AK***V*

----RER
--HEER*
----RE*
NRKMAE*
--EAEE*
-GPIPG*
-DRKAE*

*K*L****SN SNGEEGSNSD mrnssws RSFTR**I*N
IQ*L*A**D* RDNKDDI-** *KH** ..... *———******
V**L*S**TE LRSTT*S-NH RNHD*--KIC *NIK*NI*T*
LK*L*ML*DD ENSSRRC-*K SKSERLNG*R *KTMSN-V*N
*Q*L*A* *GE YYNVPEVNZ£E=32 1; ~ .2

*Q*LRS**D* ATGGGD*AA'L RRAAVPV--- RSVSCPLAI*

 

******SRDH
********E*
****Q*AQEA
*******H**
**S****DDR
*AL**TPSIA

119

Legend for Figure 4.15. The clones are from tomato (LE-ACSZ; Van Der
Straeten et al., 1990); pea (PS-ACSZ; this work); mung bean (VR-ACS1;
Botella et al., 1992); zucchini (CP-ACS1; Huang et al., 1991); winter squash
(CM-ACS1; Nakajima et al., 1990), carnation (DC-ACSl; Park et al., 1992,
modified in this work), Arabidopsis (AT-ACS1; Liang et al., 1992), and tobacco
(NT-ACS1; Bailey et al, 1992). Residues identical to those of LE-ACS3 are
shown by ”*". Gaps in the sequence are shown by ”-". The sequence of DS-
ACS1 in this work differs from the original. The original amino acid sequence
was deduced from a genomic clone. In the original sequence, a putative splice
junction that would not affect the reading frame was ignored leading to the
addition of 35 amino acid residues including an unlikely string of 18 threonine
residues. In this work, the intervening sequence was interpreted as an intron
and deleted (original position is indicated by the shaded backslashes) yielding
the sequence that matches well with this alignment.

LE-ACSZ
PS-ACSZ
VR-ACSl
CP-ACSI
CM-ACSl
DC-ACSI
AT-ACSI
NT-ACSl

LE-ACSZ
PS-ACSZ
VR-ACSl
CP-ACSl
CM-ACSl
DS-ACSl
AT-ACSl
NT-ACSl

LE-ACSZ
PS-ACSZ
VR-ACSI
CP-ACSl
CM-ACSl
DS-ACSl
AT-ACSI
NT-ACSI

LE-ACSZ
PS-ACS2
VR-ACSl
CP-ACSl
CM-ACSl
DS-ACSl
AT-ACSI
NT-ACSl

LE-ACSZ
PS-ACSZ
VR-ACSl
CP-ACSI
CM-ACSI
DS-ACSl
AT-ACSI
NT-ACSI

LE-ACSZ
PS-ACSZ
VR-ACSl
CP-ACSI
CM-ACSI
DS-ACSl
AT-ACSI
NT-ACSI

MGFEIAKT-N

S-ILSKLATN

120

EEHGENSPYF

**VMNLD--Q PQL***I*MG DG***A*S**
***KAMD--Q TPL***MAIG ac***s****

***HQIDER*
*E*HQIDER*
**SYKGVYDR
**LPGKNKGA
****NE*NSS

CLDLIEDWIK
TA*MVQN**M
TS**VE***L
SF*M*V***R
SF*M*V***R
*F***TE*LL
*****K**V*
*F***EE***

PERVVMAGGA

***I**S***
*D*I**S***
*s*1**c***
*S*I**G***
*D*I**S***
******s***
**********

FKITSKAVKE
**L*KQ*LE*
*VL*KE*LED
*QV*KA*LEI
*QV*KA*LEI
****KETLQS
**L*VD*AEW
*Q**TK*VR*

EIYAATVFDT
********SH

********SQ
***S****KA
***S****KA
**F*T***NS
********AG
********N*

VNCARKMSSF

*D*T******
**********
*RR**R****
*RR**Q****
*ST**R****
*s********
**********

QAL***I*LD
QAL***I*VD
-E****I***
--v***1***
--********

RNPKGSICS-
S**EA***TL
N**EA***TP
KH*EA***TP
KH*EA***TP
K**QA***TN
E**EA***T*
***NA***TT

TGANETIIFC
***H*ATA**
***H*VTA**
***s**v***

***s**v***
SAS-**LL**
*******M**
**********

AYENAQKSNI
***K*KED**
***K*RED**
**KK**EA*M
*tKK**EA**
***EL*-K**
**KK*****K
**********

PQFVSIAEIL
*s*1*****1
*G*I******
*T*T*****V
*T*I***Q*V
*s*1*v**v1
cp***v**vv
**********

GLVSTQTQYF

*********L
*********L
****S***HL
****S***HL
****S***FM
****S***LM
*********L

DG********
DG********
DG****LE**
NQ*****E**
**L*******

EGIKSFKAIA
**VHN**QM*
***ND*RA**
*tLER**S**
K*LER**S**
**VNK*MD**
***HQ*SD**

******R***

LADPGDAFLV
***R*E*L**
*****E****
**********
**********
**N******I
******v**1
***T******

KVKGLILTNP
RF***LI***
R****LI***
****v*1***
****v*1***
****I*V***
**********
**********

DEQEMTYCNK
EHDTDIE*DR
EDETDIE*DR
EQM*H--*K*
E*M*H--*K*
KDMPHV--*Q
NDVDISEV*V
*DE*SH-***

LAAMPSDEKF
I*K*L**DD*
**S*LN*DE*
****L***D*
****L***D*
I**LL**DD*
L*S*L**DQ*
**E*L***R*

DGWKAYDSDP
*******K**

*******QN*
*******N**
*******N**
*******R**
*******K**
*******N**

NFQDYHGLPE
**********

********A*
**********
**********
I*********
********KK
**********

PSPYYPAFNR
*T****G*D*
*I****G*D*
*****Ag*p*
*****A**D*
********N*
*****A**D*
**********

SNPLGTTLDK
******VM*R

******IM*R
*******y*R
*******Y*R
******v***
******M***
**********

DLVHIVYSLS
n*********

u*********
E*I**L****
E*I**L****
*****L****
**I*******
**********

VDNFLRESAM
*EK**A***K
*ER**A***K
**K**A*NSK
**K**A*NSK
*RR**V**RD
*****M**SR
*S***T**SK

FHPLKNPHGV
***s******

***TD**N**
***EN**L**
***ED**L**
Y*ST**SN**
*tLSR****I
*******N**

FRKAIAKFME
**N*V****S
**N*V****A
**N***N**G
**NG**S**G
**s*v****g
**Q***HG*G
**s*******

DLRWRTGVQL

********K*
********K*
**K***RAQI
**K***RAQI
********N*
********EI
********Q*

DTLKSVLSFT
N**RT*IT*I
K**RT*V**I
****TLVT*V
****TLVT*V
****ML*T*V
***TNLVR*V
****NL*T**

KDMGLPGFRV
****F*****

****F*****
**********
**********
****M*****
**********
**********

RLGKRHKHFT
**AQ*YRV**
**AQ*FRV**
*V*E**AR**
**AE**AR**
**FR**Q***
***I***V**
**A*******

IQMGLAENQL

**********
M*********
**********
**********
**********
**********
**********

KTRGGRVRFD
R***N**T**
R***N*IT**
*V*****K**
*V*****Q**
*A*DEK*I*N
*A*****T**
*******T**

IPIHCESSNN
v*v1******
V*VM*D****
*RV**NG***
*RV**N****
**FT*S****
**VP*S**D*
***p*D****

NQHNIHLVCD
*EKR***IS*
*EKR******
***D***I**
***D***I**
*AK*******

TRK*****V*
**********

GIIYSFNDDV
*****y**T*

*****y**3*
*****y**v*
*****y**v*
*****y**R*
**v*****s*
**V*****A*

NGLEVVGIKC

G**IK*****
G**AK*****
KE*DKM**T*
KE*DKM**T*
SE*AKI**G*
T*IKKAD*A*
****E*****

Figure 4.15. Amino acid sequence comparison of ACC synthase clones similar
to LE-ACSZ.

Figure 4.1 5 (continued)

LE-ACSZ
PS-ACSZ
VR-ACSI
CP-ACSI
CM-ACSl
DS-ACSl
AT-ACSI
NT-ACSI

LE-ACSZ
PS-ACSZ
VR-ACSl
CP-ACSl
CM-ACSl
DS-ACSl
AT-ACSl
NT-ACSl

LE-ACSZ
PS-ACSZ
VR-ACSI
CP-ACSl
CM-ACSI
DS-ACSl
AT-ACSl
NT-ACSI

LKNNAGLFCW MDLRPLLRE-

121

STFDSEMSLW RVIINDVKLN VSLGSSFECQ EPGWFRVCFA

 

 

*QS*G***V* ****G**KN- A**E**IE** ****HE**I* **p*v**a*s
*QS*****V* ****Q**KKP P*****TE** ****HE**I* **p*y**H*T
aus***v*v* ****R**KD-'Q**KA**E** *****E**** **P****HVT
*NS***V*V* ****R**KD- Q'ktm'k'kEtir *****E**** **P****HV'1‘
*QG**A**V* ****H**D*-A*VER*LK** *****E**I* **P****L*S
*TS*****A* ****H***DR NS*E**IE** HI**DR**** **P****R*T
*RS******* *******K*- ********** ********** **p****p**
NMDDGTVDIA LARIRRFV-- GVEKSGDKSS SHE ------- -KKQQWKKNN
****RD*Q** *Q***s**——.TQNNKEAMGJ DK ........ NS*PY*-HS*
****MA*Q** *Q***N**-'LQN*EVVVS- .......... N**HC*-HS*
****N***V* *N**HS**-'ENIDKKEDNT‘VAMPS ------- *TRHRD*K
****N***V* *N**HS**--'ENIDKKEDNT'VAMPS ------- *TRRRE*K
***NA*L*V* *N***S**--'TRGR-IP*53; ———— *K'RGQME
****D*LHV* *G**QD**SK.N KN*IVE*Afi ENDQVIQNKS.AK*LK*TQT*
****E***** *****s**-- **K****E*T PILME ..... -K********
RMYVLDES ------ SPLS-S PIPPSPLVR

*FDDIMM ....... *wH--* ***Q****KA TT

*FDDITM ------- **H--* *L*Q**M*KA TN

*YDEGNVLN- ----**HIM* *--H****IA KN

*YDEGNVLN- ----**HTM* *--H****IA KN

*FEDGLM*PH SILL**H--* *M*Q****KA RT

*L*EDGL* ------ **GIM* *--H***L*A

***DESVNL- ___-****-* ***H*****A RT

********y*
********y*
**********
**********
**********
******I***
***F******

LRLSFS---K
****LKP--R
****LKT--R
******FSGR
******FSGR

*****NN--R
*****----R

******—-—*

122
Of all the ACC synthases, the LE-ACSZ group appears to be induced by

the widest range of stimuli. Although other groups such as those related to LE-
ACS4 (Olson et al., 1991; Rottman et al., 1991) show significant similarity to
the LE-ACSZ-like group, members of the LE-ACSZ group can be distinguished
from the rest by the residues Ser-Pro repeated three times at the C—terminus.
This group seems to represent the primary transcript in ripening fruits of
tomatoes (Olson et al., 1991; Rottman et al., 1991) and zucchini (Huang et al.,
1991). In Arabidopsis, it is expressed in all light-grown tissues examined and
in etiolated seedlings (Liang et al., 1992). It is present at low levels and
increases following auxin treatment in Arabidopsis (Liang et al., 1992), zucchini
(Huang et al., 1991), mung bean (Botella et al., 1992), apple (Kim et al.,
1992), and tomato (Yip et al., 1992). Its expression is increased by wounding
in Arabidopsis seedlings (Liang et al., 1992), tomato fruit (Olson et al., 1991;
Rottman et al., 1991; Yip et al., 1992), and zucchini hypocotyls (Huang et al.,
1991). Flooding in tomato roots (Olson et al., 1995) and pathogen infection
in tomato leaves or suspension cells (Spanu et al., 1993) also cause the
transcript to accumulate. It also has been proposed that VR-ACS1 is induced
by touch (Botella et al., 1995).

The diversity of stimuli that induce expression of the PS-ACSZ family
suggests surprisingly complex regulation. Six to nine genes for ACC synthase
are thought to be expressed in tomato (Zarembinski and Theologis, 1994). If

ACC synthase is encoded by a multi-gene family whose members are induced

123

under different conditions in different tissues, why is there a member that
appears to be induced under so many conditions? One possibility is that this
group represents the earliest ACC synthase which was originally responsible for
all ethylene production. The rest of the gene family may have evolved from
gene duplication and diverged to fill more specific roles. In support of this
hypothesis, a PCR fragment for an ACC synthase from Marsilea quadrifolia, a
semi-aquatic fern, has been isolated (J. Chernys, personal communication), and
it shows the greatest similarity to the PS-ACSZ-type group. The C-terminal
sequence will have to be determined to verify whether it contains the Ser-Pro
repeat characteristic of this group.

The IAA-induced accumulation of transcripts for PS-ACS1 and PS-ACSZ
was repressed in the presence of ethylene. The repression by ethylene is
consistent with previous studies which showed that ethylene negatively

regulates its own biosynthesis in vegetative tissues (Kende, 1993).

124
REFERENCES

Abel S, Nguyen MD, Chow W, Theologis A (1995) ACS4, a primary
indoleacetic acid-responsive gene encoding 1-aminocyclopropane-1-
carboxylate synthase in Arabidopsis thaliana. J Biol Chem 270: 19093-
19099

Abeles FB, Morgan PW, Saltveit ME (1992) Ethylene in Plant Biology.
Academic Press, New York

Botella JR, Arteca JM, Schlagnhaufer CD, Arteca RN (1992) Identification and
characterization of a full-length cDNA encoding for an auxin-induced 1-
aminocyclopropane-1-carboxylate synthase from etiolated mung bean
hypocotyl segments and expression of its mRNA in response to indole-3-
acetic acid. Plant Mol Biol 20: 425-436

Botella JR, Arteca RN, Frangos JA (1995) A mechanical strain-induced 1-
aminocyclopropane-1 -carboxylic acid synthase gene. Proc Natl Acad Sci
USA 92: 1595-1598

Czarnecka E, Nagao R, Key JL, Gurley WB (1988) Mol Cell Biol 8: 1113-1122

Destafano-Beltran LJC, Caeneghem WV, Gielen J, Richard L, Van Montagu M,
Van Der Straeten (1995) Characterization of three members of the ACC
synthase family in Solanum tuberosum L. Mol Gen Genet 246: 496-508

Dong JG, Kim WT, Yip WK, Thompson GA, Li L, Bennett AB, Yang SF (1991)
Cloning of a cDNA encoding 1-aminocyclopropane-1-carboxylate
synthase and expression of its mRNA in ripening apple fruit. Planta 185:
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CONCLUSIONS

From the data presented in this work, a model of how positive and negative
feedback are involved the regulation of ethylene biosynthesis is proposed (Figure 5.1).
A stimulus, IAA in the present work, causes an increase in ACC synthase transcript
abundance leading to an increase in ACC synthase activity. The newly formed ACC is
converted to ethylene by a low, constitutive level of ACC oxidase. The ethylene
produced then causes an increase in the levels of ACC oxidase transcript and activity via
a positive feedback loop. Ethylene also causes a decrease in ACC synthase transcript and
activity via a negative feedback loop. Although the sequential induction of the ethylene
biosynthetic enzymes has not been shown in other plants, results from studies on wound-
induced ethylene production in mung bean hypocotyls (Kim and Yang, 1994) and in
winter squash mesocarp (Hyodo et al. , 1993) indicate that the model could apply to the
regulation of ethylene formation by other stimuli.

At first, the positive and negative feedback loops would appear contradictory.
The increase in ACC oxidase would presumably enhance ethylene production while the
repression of ACC synthase would decrease ethylene production. However, this apparent
contradiction arises from considering these two events to be simultaneous. The increase
in ACC oxidase may precede the negative feedback on ACC synthase. This temporal
separation could arise from the kinetics of different signal transduction pathways.

Alternatively, the positive feedback on ACC oxidase may be initiated by a lower ethylene

127

Methionine .

SAM Synthetase l

SAM
ACC Synthase T <

 

ACC
ACC Oxidase 1+ <

 

 

Ethylene .................. __

Figure 5.1. Positive and negative feedback of ethylene on the biosynthetic
pathway after auxin treatment.

128

129
concentration than that required by the negative feedback on ACC synthase. These

possibilities need to be examined to understand the roles of the two feedback loops.

Like ACC synthase, ACC oxidase is also encoded by a multigene family (for a
review, see Zarembinski and Theologis, 1994). Because the coding region of ACC
oxidase is very highly conserved, it is likely that the probe (PE8) used in the current
work would hybridize to transcripts of any ACC oxidase genes that were expressed.
Only clones related to PE8 were isolated during the library screening. However, the
library was made from tissue that would be enriched for an ethylene-inducible ACC
oxidase. A sufficient number of clones were not isolated to ensure that a lower
abundance constitutive ACC oxdidase would be isolated. Thus, it is not clear whether
PE8 is both constitutively expressed and ethylene-inducible or whether different genes
are responsible for the two types of expression. Analysis using 3’-UTRs should be able
to answer this question.

The role of ACC oxidase in ethylene-insensitive mutants of Arabidopsis, such as
err] , is an interesting problem. The level of ACC oxidase seems to correspond to the
level of endogenous ethylene (Schierle et al., 1989; Hyodo et al. , 1993; Kim and Yang,
1994; Peck and Kende, 1995). Thus, the level of ACC oxidase should be quite low in
insensitive mutants. Although ACC oxidase activity has never been measured directly,
it has been shown that the rate of ethylene production in even the most severe mutants
is actually higher than in wild type (Bleecker et al., 1988; J. Ecker, personal
communication). Is a low level of constitutive ACC oxidase sufficient to maintain

normal ethylene production? The fact that ethylene production is reduced by about 70%

130

when the ACC oxidase activity is decreased by 70% in leaves of transgenic tomato plants
expressing antisense ACC oxidase would argue that this is not the case (Hamilton et a1. ,
1990). These results indicate that the positive feedback of ethylene on ACC oxidase may
utilize a signal transduction pathway different from the one involved in the formation of

the apical hook.

131

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