Characterization of the 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase multigene family of Malus domestica Borkh

Article abstract of DOI:10.1016/j.phytochem.2009.01.002

Two 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACO) genes have been cloned from RNA isolated from leaf tissue of apple (Malus domestica cv. Royal Gala). The genes, designated MD-ACO2 (with an ORF of 990 bp) and MD-ACO3 (966 bp) have been compared with a previously cloned gene of apple, MD-ACO1 (with an ORF of 942 bp). MD-ACO1 and MD-ACO2 share a close nucleotide sequence identity of 93.9% in the ORF but diverge in the 3′ untranslated regions (3′-UTR) (69.5%). In contrast, MD-ACO3 shares a lower sequence identity with both MD-ACO1 (78.5%) and MD-ACO2 (77.8%) in the ORF, and 68.4% (MD-ACO1) and 71% (MD-ACO2) in the 3′-UTR. Southern analysis confirmed that MD-ACO3 is encoded by a distinct gene, but the distinction between MD-ACO1 and MD-ACO2 is not as definitive. Gene expression analysis has shown that MD-ACO1 is restricted to fruit tissues, with optimal expression in ripening fruit, MD-ACO2 expression occurs more predominantly in younger fruit tissue, with some expression in young leaf tissue, while MD-ACO3 is expressed predominantly in young and mature leaf tissue, with less expression in young fruit tissue and least expression in ripening fruit. Protein accumulation studies using western analysis with specific antibodies raised to recombinant MD-ACO1 and MD-ACO3 produced in E. coli confirmed the accumulation of MD-ACO1 in mature fruit, and an absence of accumulation in leaf tissue. In contrast, MD-ACO3 accumulation occurred in younger leaf tissue, and in younger fruit tissue. Further, the expression of MD-ACO3 and accumulation of MD-ACO3 in leaf tissue is linked to fruit longevity. Analysis of the kinetic properties of the three apple ACOs using recombinant enzymes produced in E. coli revealed apparent Michaelis constants (K_m) of 89.39 μM (MD-ACO1), 401.03 μM (MD-ACO2) and 244.5 μM (MD-ACO3) for the substrate ACC, catalytic constants (K_cat) of 6.6 × 10^-2 (MD-ACO1), 3.44 × 10^-2 (Md-ACO2) and 9.14 × 10^-2 (MD-ACO3) and K_cat/K_m (μM s^-1) values of 7.38 × 10^-4 μM s^-1 (MD-ACO1), 0.86 × 10^-4 M s^-1 (MD-ACO2) and 3.8 × 10^-4 μM s^-1 (MD-ACO3). These results show that MD-ACO1, MD-ACO2 and MD-ACO3 are differentially expressed in apple fruit and leaf tissue, an expression pattern that is supported by some variation in kinetic properties.

Full text of DOI:10.1016/j.phytochem.2009.01.002

Phytochemistry 70 (2009) 348–360
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Characterization of the 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase
multigene family of Malus domestica Borkh
*
Jan E. Binnie, Michael T. McManus
Institute of Molecular Biosciences, Massey University, Private Bag 11222, Palmerston North, New Zealand
a r t i c l e i n f o
a b s t r a c t
Article history:
Two 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACO) genes have been cloned from RNA iso-
lated from leaf tissue of apple (Malus domestica cv. Royal Gala). The genes, designated MD-ACO2 (with an
ORF of 990 bp) and MD-ACO3 (966 bp) have been compared with a previously cloned gene of apple, MD-
ACO1 (with an ORF of 942 bp). MD-ACO1 and MD-ACO2 share a close nucleotide sequence identity of
93.9% in the ORF but diverge in the 30 untranslated regions (30-UTR) (69.5%). In contrast, MD-ACO3 shares
a lower sequence identity with both MD-ACO1 (78.5%) and MD-ACO2 (77.8%) in the ORF, and 68.4% (MD-
ACO1) and 71% (MD-ACO2) in the 30-UTR. Southern analysis confirmed that MD-ACO3 is encoded by a dis-
tinct gene, but the distinction between MD-ACO1 and MD-ACO2 is not as definitive. Gene expression anal-
ysis has shown that MD-ACO1 is restricted to fruit tissues, with optimal expression in ripening fruit, MD-
ACO2 expression occurs more predominantly in younger fruit tissue, with some expression in young leaf
tissue, while MD-ACO3 is expressed predominantly in young and mature leaf tissue, with less expression
in young fruit tissue and least expression in ripening fruit. Protein accumulation studies using western
analysis with specific antibodies raised to recombinant MD-ACO1 and MD-ACO3 produced in E. coli con-
firmed the accumulation of MD-ACO1 in mature fruit, and an absence of accumulation in leaf tissue. In
contrast, MD-ACO3 accumulation occurred in younger leaf tissue, and in younger fruit tissue. Further,
the expression of MD-ACO3 and accumulation of MD-ACO3 in leaf tissue is linked to fruit longevity. Anal-
ysis of the kinetic properties of the three apple ACOs using recombinant enzymes produced in E. coli
Received 8 November 2008
Received in revised form 19 December 2008
Available online 14 February 2009
Keywords:
Malus domestica
ACC oxidase
Apple fruit development
Apple leaf development
Ethylene
revealed apparent Michaelis constants (Km
) of 89.39 lM (MD-ACO1), 401.03 lM (MD-ACO2) and
244.5
l
M
(MD-ACO3) for the substrate ACC, catalytic constants (Kcat
)
of 6.6 ꢀ 10ꢁ2 (MD-ACO1),
3.44 ꢀ 10ꢁ2 (Md-ACO2) and 9.14 ꢀ 10ꢁ2 (MD-ACO3) and Kcat/Km
(l
M sꢁ1) values of 7.38 ꢀ 10ꢁ4
l
M sꢁ1
(MD-ACO1), 0.86 ꢀ 10ꢁ4 M sꢁ1 (MD-ACO2) and 3.8 ꢀ 10ꢁ4
l
M sꢁ1 (MD-ACO3). These results show that
MD-ACO1, MD-ACO2 and MD-ACO3 are differentially expressed in apple fruit and leaf tissue, an expression
pattern that is supported by some variation in kinetic properties.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
a reaction that proceeds in the presence of O2 (Ververidis and John,
1991; Bouzayen et al., 1991; Dong et al., 1992a,b). In many studies
Ethylene is a gaseous plant hormone that regulates many pro-
cesses associated with plant growth and development (Abeles
et al., 1992). In higher plants ethylene is synthesized via two com-
published thus far, ACS is considered the rate-determining step in
the committed ethylene biosynthetic pathway, and in all plant spe-
cies examined in any detail thus far, ACS is comprised of a larger
gene family. Indeed, in Arabidopsis, 12 genes have been identified
in the family. In contrast, ACO has been characterised as a smaller
differentially expressed multigene family in many plant species
including tomato (Bouzayen et al., 1993; Barry et al., 1996; Nakat-
suka et al., 1998), peach (Ruperti et al., 2001; Rasori et al., 2003),
melon (Lasserre et al., 1996, 1997) and in white clover (Hunter
et al., 1999; Chen and McManus, 2006).
For apple (Malus domestica Borkh), the first ACO gene identified
was designated MD-ACO1 [clone pAP4, (Ross et al., 1992); clone
pAE12, (Dong et al., 1992a,b)]. Subsequently, three further MD-
ACO genes have been identified (Wiersma et al., 2007). MD-ACO1
expression and its role in fruit ripening has been a major research
mitted enzyme-catalyzed steps from S-adenosyl-L-methionine
(SAM). The first of these, 1-aminocyclopropane-1-carboxylic acid
(ACC) synthase (ACS) catalyzes the cyclization of SAM to ACC and
subsequently ACC oxidase (ACO) catalyzes the oxidative conver-
sion of ACC to ethylene (Adams and Yang, 1979; Yang and
Hoffman, 1984; Kende, 1993). ACS is a pyroxidal phosphate-requir-
ing enzyme while ACO is a non-haem iron enzyme which requires
Fe2+ and CO2 as co-factors as well as ascorbate as a co-substrate in
* Corresponding author. Tel.: +64 6 356 9099; fax: +64 6 350 5688.
E-mail address: M.T.McManus@massey.ac.nz (M.T. McManus).
0031-9422/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2009.01.002

J.E. Binnie, M.T. McManus / Phytochemistry 70 (2009) 348–360
349
focus for many years in apple, including the analysis of the gene
promoter and the phenotypes of anti-sensed ACO plants (Dong
et al., 1992a,b; Ross et al., 1992; Dilley et al., 1995; Zhu et al.,
1995; Bolitho et al., 1997; Atkinson et al., 1998; Tan and Bangerth,
2000; Costa et al., 2005; Cin et al., 2005; Park et al., 2006). Further,
ACO activity has been extracted from apple fruit tissue and the en-
zyme extensively characterized both as a purified protein (Dong
et al.,1992a,b; Fernández-Maculet and Yang, 1992; Kuai and Dilley,
1992; Poneleit and Dilley, 1993; Fernández-Maculet et al., 1993;
Pirrung et al., 1993; Dilley et al., 1993; Dupille et al., 1993;
Mizutani et al., 1995) and as a recombinant protein (Charng
et al., 1997; Charng et al., 2001; Yoo et al., 2006).
The focus on MD-ACO1 and the coded enzyme reflects an inten-
sive research interest into the role of ethylene in fruit ripening and
for apple, particularly. However, given the evidence of differential
expression of ACO multigene families in many other plant species,
and the realisation that a small multi-gene family is also present in
apple (Wiersma et al., 2007), there are no studies on the expression
of other members of the gene family or biochemical characteriza-
tion. Further, ethylene plays an important role in leaf development
and, as yet, there have been fewer studies that focus on the differ-
ential expression of ACO genes during leaf ontogeny (Hunter et al.,
1999; Chen and McManus, 2006).
gions of PP-ACO1 and PP-ACO2 (77.7%) is higher than for the 50
and 30-UTRs of 44% and 50.4%, respectively (Ruperti et al., 2001).
Identities between the MD-ACO amino acid sequences broadly
reflected the percentage identities observed for the nucleotide cod-
ing sequences with highest identity observed between MD-ACO1
and MD-ACO2 (92.04%), with less identity between MD-ACO1 and
MD-ACO3 (80.57%) and MD-ACO2 and MD-ACO3 (75.16).
2.2. Evidence of a multi-gene family in apple by genomic Southern
analysis
DNA isolated from leaves of apple was digested with either
EcoRI, HindIII or XbaI, and probed with sequences comprising the
30 region of the coding sequence and a contiguous region of the
3’-UTR to generate gene-specific probes. The Southern analysis
demonstrates that the MD-ACO3 sequence is encoded by a gene
distinct from both MD-ACO1 and MD-ACO2, as the hybridization
patterns following restriction digests with EcoRI, HindIII and XbaI,
clearly differ (Fig. 1). However, each one of the restriction digested
fragments which hybridises with the MD-ACO2 probe are of the
same approximate size as fragments which hybridize with the
MD-ACO1 probe, although the relative intensity of many of the
bands differs in each digest when probed with either MD-ACO1
or MD-ACO2. An example can be seen in the HindIII digest, where
fragments of 1.5 and 1.9 kb strongly hybridize with MD-ACO1 com-
pared with the one fragment of 1.9 kb which hybridizes with MD-
ACO2 (Fig. 1).
In this paper, therefore, we aim to isolate expressed ACO genes
from apple leaf tissue, to examine their expression during leaf and
fruit ontogeny in apple as well as to functionally express these MD-
ACO cDNAs in E. coli to characterize each gene product,
biochemically.
2.3. Analysis of thededuced translational sequences of each of the ACO
proteins
2. Results
From a deduced translation of each of the three ACO open read-
ing frames (ORFs), amino acid sequences were aligned and com-
pared (Fig. 2). From previous studies with recombinant protein
2.1. Nucleotide and deduced amino acid sequence comparison of
MD-ACO1, MD-ACO2 and MD-ACO3
RT-PCR approaches, using RNA isolated from developing, ma-
ture-green and senescent leaf tissue of apple, amplified cDNAs of
ca. 850 bp. Subsequent sequencing revealed that two distinct
ACO sequences were identified from RNA isolated from mature-
green leaf tissue (designated MD-ACO2 and MD-ACO3), which were
also distinct from MD-ACO1. All three MD-ACO sequences were
then aligned against an apple EST library maintained at the Horti-
cultural and Food Research Institute of New Zealand (HortRe-
search). The relevant ESTs are now publicly available on Genbank
with the accession numbers of MD-ACO1 (X61390), MD-ACO2
(EB14037) and MD-ACO3 (EB151006). Further, an alignment, at
the nucleotide level, of the genes identified in this study (and
verified in the HortResearch EST library) with those reported by
Wiersma et al. (2007) show that MD-ACO2 most closely aligns
(98%) with MD-ACO2 (AF015787) reported in Wiersma et al
(2007) an MD-ACO3 is equivalent (99% identity) with MD-ACO3
(AF086888) reported in Wiersma et al (2007).
Comparison of nucleotide sequences of the putative MD-ACO
multi-gene family revealed that the coding sequences of MD-
ACO1 and MD-ACO2 are quite highly identical (93.9%) with less
identity between MD-ACO1 and MD-ACO3 (78.5%) and MD-ACO2
´
and MD-ACO3 (77.8%). When the 3-UTR sequences are compared,
then the degree of identity between MD-ACO1 and MD-ACO2 de-
creases (69.5%) while yet lower identities are observed between
MD-ACO1 and MD-ACO3 (68.4%) and MD-ACO2 and MD-ACO3
(71%). The observation of lower identities between the 30-UTR se-
quences when compared with the coding sequences of ACO mul-
ti-gene families is consistent with other studies (e.g., white
clover) where greater identity was found over the coding region
(>90%) when compared with the 30-UTR (ꢂ70%) (Hunter et al.,
1999). Likewise, in peach, where identity between the coding re-
Fig. 1. Southern analysis of Malus domestica genomic DNA. The hybridisation
pattern of 32P labelled MD-ACO1, 32P labelled MD-ACO2, 32P labelled MD-ACO3, as
indicated, with genomic DNA digested with EcoRI (E), HindIII (H) and XbaI (X).
Molecular sizes are indicated.

350
J.E. Binnie, M.T. McManus / Phytochemistry 70 (2009) 348–360
MD-ACO1
MD-ACO2
MD-ACO3
M A T F P V V D L S L V N G E E R A A T L E K I N D A C E N
M A T F P V V D M D L I N G E E R A A T L E K I N D A C E N
M E N F P V I N L E S L N G E G R K A T M E K I K D A C E N
28
MD-ACO1
MD-ACO2
MD-ACO3
W G F F E L V N H G M S T E L L D T V E K M T K D H Y K K T
W G F F E L V N H G I S T E L L D T V E K M N K D H Y K K T
W G F F E L V S H G I P T E F L D T V E R L T K E H Y K Q C
60
MD-ACO1
MD-ACO2
MD-ACO3
M E Q R F K E M V A A K G L D D V Q S E I H D L D W E S T F
M E Q R F K E M V A A K G L E A V Q S E I H D L D W E S T F
L E Q R F K E L V A S K G L E G V Q T E V K D M D W E S T F
72
MD-ACO1
MD-ACO2
MD-ACO3
F L R H L P S S N I S E I P D L E E E Y R K T M K E F A V E
F L R H L P S S N I S E I P D L E E D Y R K T M K E F A V E
H L R H L P Q S N I S E V P D L K D E Y R N V M K E F A L K
MD-ACO1
MD-ACO2
MD-ACO3
L E K L A E K L L D L L C E N L G L E K G Y L K K V F Y G S
L E K L A E K L L D L L C E N L G L E K G Y L K K A F Y G S
L E K L A E Q L L D L L C E N L G L E Q G Y L K K A F Y G T
133
144
MD-ACO1
MD-ACO2
MD-ACO3
K G P N F G T K V S N Y P P C P K P D L I K G L R A H S D A
K G P N F G T K V S N Y P P C P K P D L I K G L R A H T D A
K G P T F G T K V S N Y P P C P N P D L I K G L R A H T D A
157 158
165
172
175 177 179
MD-ACO1
MD-ACO2
MD-ACO3
G G I I L L F Q D D K V S G L Q L L K D G E W V D V P P M H
G G I I L L F Q D D K V S G L Q L L K D G E W M D V P P V H
G G L I L L F Q D D K V S G L Q L L K D G E W V D V P P M R
199
MD-ACO1
MD-ACO2
MD-ACO3
H S I V I N L G D Q I E V I T N G K Y K S V M H R V I A Q S
H S I V I N L G D Q I E V I T N G K Y K S I M H R V I A Q S
H S I V I N L G D Q L E V I T N G K Y K S V E H R V I A Q T
230
234
MD-ACO1
MD-ACO2
MD-ACO3
D G T R M S I A S F Y N P G N D S F I S P A P A V L E K K T
D G T R M S I A S S Y N P G D D A F I S P A P A L L E K K S
D G T R M S I A S F Y N P G S D A V I Y P A P T L V E K E A
244 246
MD-ACO1
MD-ACO2
MD-ACO3
E D
P T Y P K F V F D D Y M K L Y S G L K F Q A K E P R F
E E T P T Y P K F L F D D Y M K L Y S G L K F Q A K E P R F
E E K N Q V Y P K F V F E D Y M K L Y A G V
K
F E A K E P R
292
296 297 299
MD-ACO1
MD-ACO2
MD-ACO3
E A M K A K E S T P V A T A
E A M K A R E T T P V E T A R G L R A V R W N T T K R N Q N
F E A M K A V E I K A S F G L G P V I S T A
301
Fig. 2. Comparison of the deduced amino acid sequences of the open reading frames of MD-ACO1, MD-ACO2 and MD-ACO3, as indicated. Amino acids important for apple ACO
activity are in bold, and numbered, and the conserved lysine and cysteine residues are italicized, and numbered.
expressed from clones pAP4 and pAE12 (equivalent to MD-ACO1 in
this study), activity requires that some specific amino acid residues
be conserved. Specifically, residues H177, D179 and H234 are required
to bind iron in the active site (Shaw et al., 1996; Kadyrzhanova
et al., 1997; Kadyrzhanova et al., 1999), while R244, S246 and T157
as binding sites for the ACC carboxyl group (Kadyrzhanova et al.,
1999; Dilley et al., 2003; Seo et al., 2004). Also, the lysine residues
bate binding. In the C-terminus, E297, R299 and E301 (E298, R300 and
E302 in MD-ACO3) are important for enzyme activity, particularly
R299 (R300 in MD-ACO3) which may be involved in the mechanism
of CO2 activation (Kadyrzhanova et al., 1999). Three conserved cys-
teine residues C28, C133 and C165 have been observed of which the
C28 residue only is important for catalysis (Kadyrzhanova et al.,
1999; Dilley et al., 2003). In common with MD-ACO1, the two other
sequences, MD-ACO2 and MD-ACO3 also contain the specific resi-
dues outlined. Further, MD-ACO3 contains a fourth cysteine resi-
due at position 60 (C60).
K
158, K230 or K292 (K293 in MD-ACO3) are strongly implicated for
ascorbate activation (Kadyrzhanova et al., 1999; Dilley et al.,
2003) and residue R175 has been proposed to bind directly to NaH-
CO3 (Dilley et al., 2003). Eight lysine residues [K72, K144, K158, K172
K
,
199, K230, K292 and K296 (K293 and K297 in MD-ACO3)] observed in
2.4. Confirmation of MD-ACO differential gene expression in vivo
pAP4 and pAE12 are conserved in other plant species (Kadyrzhano-
va et al., 1999; Dilley et al., 2003), and in common with K158, K230
Semi-quantitative (sq)RT-PCR analysis of MD-ACO1, MD-ACO2
and MD-ACO3 gene expression is shown as Fig. 3, and the amplifi-
cation products of the expected sizes are indicated for ribosomal
or K292 (K293 in MD-ACO3), the other five lysine residues [K72, K144
,
K
172, K199, K296 (K297 in MD-ACO3)] may also be involved in ascor-

J.E. Binnie, M.T. McManus / Phytochemistry 70 (2009) 348–360
351
Fig. 3. Semi-quantitative (sq)RT-PCR analysis of MD-ACO1, MD-ACO2 and MD-ACO3 expression in vivo, as indicated. RNA was extracted from pooled young leaf (YL), mature
leaf (ML), young fruit (YF) and mature fruit (MF) tissues, harvested from 15 trees, as indicated. The molecular weights (1 Kb DNA ladder, Gibco BRL) of two of the standards are
indicated on the left of the figure. The approximate size of the amplification products are indicated by arrows to the right of the figure (18S ribosomal RNA ca. 315 bp; putative
MD-ACOs ca. 790–800 bp). The PCR products were visualized using ethidium bromide staining.
RNA (315 bp), MD-ACO1 (800 bp), MD-ACO2 (790 bp) and MD-ACO3
(800 bp). Primers specific for MD-ACO1 amplified products from
mature apple fruit tissue (17 WAFB) and less intensely from young
apple fruit tissue (7 WAFB), with no visible band for either young
or mature green leaf tissue. By contrast, primers specific for MD-
ACO2 amplified products from both apple leaf and fruit tissue,
but with a greater intensity in the fruit tissue, whilst MD-ACO3
gene-specific primers amplified products in all tissues, although
less intensely in tissue from young fruit and less again from mature
fruit. No expression, using (sq)RT-PCR, was determined in senes-
cent leaf tissue (data not shown).
recognised protein predominantly in the mature apple fruit extract
(Fig. 5; lane 3). This is consistent with the gene expression studies
(Fig. 3) in which MD-ACO1 is expressed almost exclusively in the
mature fruit tissue. A low level of detection of gene expression is
observed in the younger fruit tissue but protein accumulation is
not detected using western analysis. This may reflect the differ-
ences in sensitivity between the two techniques or may suggest
that some post-transcriptional of the MD-ACO1 gene or posttrans-
lational modification of the MD-ACO1 protein occurs in the youn-
ger fruit tissue. In contrast, antibodies raised against MD-ACO3
recognised protein predominantly accumulating in young leaf tis-
sue (Fig. 5; lane 4), and also in young fruit and mature leaf tissue,
but to a lesser extent (Fig. 5; lanes 2 and 5). This again broadly cor-
related with the expression studies shown as Fig. 3, but did suggest
more clearly that the accumulation of the MD-ACO3 protein is
more directed towards the younger of the two tissues examined.
Again, this discrepancy between the expression data and protein
accumulation may reflect the differences in (sq)RT-PCR and wes-
tern analysis, or could suggest that some degree of post-transcrip-
tional or post-translational control is operating (although this was
not investigated directly). No detection of MD-ACO1 or MD-ACO3
was observed in senescent leaf tissue (data not shown).
2.5. Phylogenetic analysis
A phylogenetic tree constructed from an alignment of 85 ACO
sequences obtained from NCBI protein database with the deduced
amino acid sequences for MD-ACO1, MD-ACO2 and MD-ACO3 over
the entire open reading frame is shown as supplementary material
(S1) including the full name of each entry. The localised groupings
around MD-ACO1 and MD-ACO2 shown as Fig. 4A, and the group-
ings around MD-ACO3 shown as Fig. 4B. The analysis suggests that
MD-ACO1 and MD-ACO2 are distinct genes as these sequences have
split on the tree and have diverged some time ago (Fig. 4A). The
analysis also confirms that MD-ACO3 is encoded from a gene dis-
tinct as the major splits of the tree are far removed from those of
both MD-ACO1 or MD-ACO2 (Fig. 4B). MD-ACO1 and MD-ACO2 are
grouped with PP-ACO2 (peach fruit, unspecified developmental
state), while MD-ACO3 is grouped with PP-ACO1 (from softening,
ripening and wounded fruit). Interestingly, PC-ACO1 (European
pear) and JP-AOX1 and JP-AOX4 (Asian pear), which are expressed
in immature and unripe mature fruit, associate with MD-ACO1
and MD-ACO2. In contrast, PP-AOX2A, PP-AOX2B and JP-AOX3 (Asian
pear), which are expressed in ripe fruit tissue, associate with MD-
ACO3.
2.7. Seasonal expression of MD-ACO3 during apple leaf and fruit
development
The expression of MD-ACO3 and accumulation of MD-ACO3 in
leaf tissue revealed in Figs. 3 and 5 was investigated in vivo over
the summer and autumn using orchard-grown apple trees
(Fig. 6). In the example shown, MD-ACO3 was shown, using wes-
tern analysis, to accumulate in late-summer, but once fruit was
strip-picked from the sampled trees (end of March), and no further
accumulation was observed. Likewise, (sq)RT-PCR revealed that
expression of MD-ACO3 occurred in tissues in late summer, but
again after fruit was strip-picked from the trees, then expression
ceased. The detection of actin expression in these tissues post-
stripping served to confirm that mRNA was successfully isolated
from the senescent leaf developmental stages.
2.6. Protein accumulation of MD-ACO1 and MD-ACO3 in apple tissue
Western analysis was performed on extracts of developing ap-
ple leaf, mature-green, fully expanded apple leaf, young fruit (7
WAFB) and mature fruit (17 WAFB), using antibodies raised against
the gene products of MD-ACO1 and MD-ACO3 as recombinant pro-
teins. The results indicate that in apple, the accumulation of ACO is
tissue-specific, such that antibodies raised against MD-ACO1
2.8. Kinetic properties of MD-ACO1, MD-ACO2 and MD-ACO3
Each of the three apple ACO cDNAs were over-expressed in
E. coli and purified by His-tag-based affinity chromatography.

352
J.E. Binnie, M.T. McManus / Phytochemistry 70 (2009) 348–360
Fig. 4. Partial phylogenetic analysis of ACO amino acid sequences most closely associated with MD-ACO1, MD-ACO2 (A) and MD-ACO3 (C). The full phylogenetic tree with the
full name for each entry is shown as Supplementary, Fig. 1. A. P. pyrifolia (BAD61004 = PP-AOX4) mRNA from immature fruit; P. pyrifolia (BAA76387 = JP-AOX1) mRNA from
fruit; P. communis (CAA60576 = PC-ACO1) mRNA from unripe mature fruit cortical tissue; P. communis (CAH18930 = ACO1) mRNA from fruit mesocarp; Prunus persica
(AAF36484 = PP-ACO2) mRNA from fruit; P. persica (BAA96787 = PP-ACO2; partial sequence) mRNA from fruit. B. P. persica (BAA96786 = PP-ACO1; partial sequence) mRNA
from fruit; Prunus mume (Q9MB94 = ACO1) mRNA from fruit; P. persica (190934OA = ACO) mRNA from softening and wounded fruit; P. persica (CAA54449 = ACO) mRNA from
ripening fruit; P. pyrifolia (BAD61000 = JP-AOX3) mRNA from ripe fruit; P. pyrifolia (BAD60998 = PP-AOX2A) mRNA from ripe fruit; P. pyrifola (BAD60999 = PP-AOX2B) mRNA
from ripe fruit.
Fig. 5. Accumulation of MD-ACO1 and MD-ACO3 in various tissues of apple determined by Western blot analysis using antibodies raised against MD-ACO1 (A) and MD-ACO3
(B). Lane 1: Prestained low range molecular markers, lane 2: young fruit tissue (7 WAFB), lane 3: mature fruit tissue (17 WAFB), lane 4: young leaf tissue, lane 5: mature leaf
tissue. The protein extracts were made from pooled tissues harvested at the developmental stages, as indicated, from 15 trees.
However, over-expression of the recombinant MD-ACOs in E. coli
resulted in degradation of the protein as judged by fragmentation
using SDS–PAGE (data not shown). Therefore, anion exchange was
used to separate active, full-length enzyme from the inactive forms
of the protein (as well as from any other contaminating E. coli pro-
teins) and these purified recombinant enzyme preparations were
used for subsequent kinetic analysis.
Initially, the pH optima of each recombinant enzyme was deter-
mined at 1 mM ACC (Table 1) after which the optimal require-
ments of each isoform for co-substrate and cofactors were then

J.E. Binnie, M.T. McManus / Phytochemistry 70 (2009) 348–360
353
Fig. 6. (A) Changes in MD-ACO3 protein accumulation using western analysis with antibodies raised against recombinant MD-ACO3 on extracts (40
lg per lane) of pooled leaf
tissue, harvested at 11.00 am on the dates indicated, from pre-tagged bourse shoots from 15 trees. (B) Changes in MD-ACO3 gene expression using (sq)RT-PCR performed on
RNA isolated from pooled leaf tissue, harvested at 11.00 am on the dates indicated, from pre-tagged bourse shoots from 15 trees. The PCR products were visualised using a 32P
labelled gene-specific MD-ACO3 probe. The fruit was harvested on 24.03.03.
ues of 7.38 ꢀ 10ꢁ4
l
M sꢁ1 (MD-ACO1), 0.86 ꢀ 10ꢁ4
l
M sꢁ1 (MD-
ACO2) and 3.8 ꢀ 10ꢁ4
l
M sꢁ1 (MD-ACO3) were calculated (Table 2).
Table 1
Substrate and co-factor requirements for MD-ACO1, MD-ACO2 and MD-ACO3, as
In terms of thermal stability, no significant difference was found
in this study between the MD-ACO isoforms. However, the activi-
ties of the enzyme differed significantly between each of the tem-
peratures assayed over time (Table 3). ACO in apple is sensitive to
high temperature. For example, in this study the half-life (t½) at
25 °C was determined to be ꢂ29 min, at 35 °C, the t½ is ꢂ13 min
and at an incubation temperature of 45 °C, while the t½ is
ꢂ5 min 30 s at a temperature of 45 °C (Table 3).
indicated.
MD-ACO1
MD-ACO2
MD-ACO3
pH
Activity range
Optima
6.5–8.0a
7.2
6.5–8.5
7.8
6.5–8.0
7.5
Ascorbate
Optimal concentration
50% inhibition
30 mM
40 mM
30 mM
40 mM
30 mM
40 mM
FeSO4 ꢃ 7H20
3. Discussion
Optimal range
Maximum activity
P70% Inhibition
15–25
l
M
15–25
lM
15–25 lM
20
40
l
l
M
M
15
40
l
l
M
M
20
l
l
M
40
M
The characterisation of ACC oxidase gene expression, particu-
larly during apple fruit ripening, has been extensively researched,
such that the MD-ACO1 gene and the MD-ACO1 protein is perhaps
the best characterized of all of the ACC oxidases. However, in com-
mon with all other plant species examined, ACO exists as a multi-
gene family in apple, but apart from MD-ACO1, there are no studies
reported that have examined either the expression of the other
MD-ACO genes or the biochemical characterisation of their protein
products. This is the aim of this study, and to do this, RNA was ex-
tracted from leaf tissues at the developing, mature-green and
senescent leaf stage from the apple cultivar Royal Gala and degen-
erate primers used to isolate any ACC oxidase genes expressed dur-
ing these leaf developmental stages. Two additional genes were
identified, designated MD-ACO2 [EB14037 equivalent to
AF015787 in Wiersma et al. (2007)] and MD-ACO3 [EB151005
equivalent to AF086888 in Wiersam et al. (2007)]. The expression
of these genes, in addition to MD-ACO1, and the functional charac-
terization of their protein products is thus described.
Southern analysis of the cv. Royal Gala genome suggests that
two copies of MD-ACO1 occur. In previous studies, the pAP4 clone
[cv. Golden Delicious; X61390; (Ross et al., 1992); designated as
MD-ACO1 in this study] was used as a RFLP marker to construct
linkage maps of the progeny of a cross between the apple cultivars
Prima and Fiesta (in common with Royal Gala, these cultivars have
34 somatic chromosomes and a haploid number of 17), and was
found at two loci, one on chromosome 5 (pAP4a) and one on chro-
mosome 10 (pAP4b) (Maliepaard et al., 1998). Also, Southern anal-
ysis using DNA extracted from cv. Golden Delicious, and digested
with EcoRI, HindIII and BamHI and then probed with pAP4 (MD-
ACO1) (Ross et al., 1992), revealed that two fragments hybridized
with pAP4 for each of the genomic digests. Similarly, for the Golden
Delicious genomic DNA digested with EcoRI a fragment hybridised
NaþHCOꢁ3
30–40% activitya
Optimal range
Maximum activity
70–100% inhibition
0 mM
20–40 mM
30 mM
0 mM
20–40 mM
30 mM
0 mM
10–40 mM
30 mM
60 mM
60 mM
60 mM
a
Data is collected from six independent experiments.
determined at their optimal pHs and 1 mM ACC (Table 1). In terms
of co-substrate and co-factor requirements, the three isoforms
showed broadly similar requirements. In this study all MD-ACOs
were maximally activated with 30 mM NaHCO3, but were com-
pletely inhibited in the presence of 60 mM NaHCO3, were activated
by 30 mM ascorbate and were inhibited (50%) by 40 mM, and dis-
played an optimum for 15–25 lM FeSO4.
In terms of the calculation of the Michaelis-Constant (Km) and
maxium velocity (Vmax) of the MD-ACO isoforms, the activity of
MD-ACO1 approached saturation at an ACC concentration of
0.5 mM, while MD-ACO2 and MD-ACO3 were saturated at an ACC
concentration of 1 mM (Fig. 7). The apparent Km and Vmax were
determined from Lineweaver–Burk plots and these are summa-
rised in Table 2. MD-ACO2 has a lower Vmax (12.94 nmol of ethylene
mgꢁ1 minꢁ1) than the Vmax of MD-ACO1 (15.15 nmol of ethylene
mgꢁ1 minꢁ1) and MD-ACO3 (18.94 nmol of ethylene mgꢁ1 minꢁ1
)
which are similar. These values are also consistent with the Vmax
and Km values obtained using Eadie Hofstee regression (data not
shown). Further, Kcat values [ethylene evolution (mol sꢁ1)/MD-
ACO (mol)] (refer Table 2) revealed MD-ACO3 (9.14 ꢀ 10ꢁ2) to have
the higher turnover rate compared to MD-ACO1 (6.6 ꢀ 10ꢁ2) and
the very sluggish MD-ACO2 (3.44 ꢀ 10ꢁ2). The Kcat/Km
(l
M sꢁ1) is
a useful means for comparing activities between enzymes, and val-

354
J.E. Binnie, M.T. McManus / Phytochemistry 70 (2009) 348–360
16
14
12
10
8
0.2
Lineweaver-Burk Plot
0.15
0.1
0.05
0
R2 = 0.9868
6
4
2
-12.00
-7.00
4.0
-2.00
3.00
8.00
13.00
7.5 8.0 8.5 9.0 9.5
10.0
10.5
18.00
11//[[SS]]
0
0.0
1
2.5 3.0 3.5
4.5 5.0
6.0
0.5
1.5 2.0
5.5
6.5 7.0
ACC (mM)
14
13
12
11
10
9
0.8
Lineweaver-Burk Plot
8
0.6
0.4
0.2
0.0
7
6
5
R2
== 00..99998822
4
3
2
1
-5
0
5
10
6.5 7.0
15
7.5 8.0 8.5 9.0 9.5
20
10.0
1/[S]
0
0.0
1
2.5 3.0 3.5
4.5 5.0
6.0
0.5
1.5 2.0
4.0
5.5
10.5
ACC (mM)
20
18
16
14
12
10
8
Lineweaver-Burk Plot
0.35
0.3
0.25
0.2
0.15
0.1
R2 = 0.9714
6
0.05
0
4
2
-5
0
5
10
15
20
1//[[SS]]
0
0.0
1
2.5 3.0 3.5
4.5 5.0
6.0
7.5 8.0 8.5 9.0 9.5
10.0
0.5
1.5 2.0
4.0
5.5
6.5 7.0
10.5
ACC (mM)
Fig. 7. Recombinant MD-ACO1 (A), MD-ACO2 (B) and MD-ACO3 (C) activity as a function of ACC concentration. Each inset is the corresponding Lineweaver–Burk (double
reciprocal) plot. Values and means SE (n = 6).
strongly with pAP4 of ca. 2.5 kb and less intensely with a fragment
of ca. 5.0 kb.
may either be tightly clustered on the genome and encoded by dis-
tinct genes, or they are alleles of the same gene.
By contrast one copy of MD-ACO2 appears to be present in the
cv. Royal Gala genome. However, it is also interesting to observe
that the restriction digested fragments which have hybridized with
MD-ACO2 are the same approximate size as fragments which have
hybridized with MD-ACO1 suggesting that MD-ACO1 and MD-ACO2
From the Southern analysis performed, MD-ACO3 is clearly a
gene distinct from both MD-ACO1 and MD-ACO2. Further, MD-
ACO3 is probably present in the cv. Royal Gala genome as a single
copy, but the two fragments which hybridize with the probe fol-
lowing the HindIII digest also raises the possibility that two copies

J.E. Binnie, M.T. McManus / Phytochemistry 70 (2009) 348–360
355
Table 2
results of this study neither MD-ACOs have been observed to fulfil
this role in leaf tissue.
Summary of kinetic properties of MD-ACO1, MD-ACO2 and MD-ACO3.
In addition to MD-ACO1, neither MD-ACO2 nor MD-ACO3 appear
to be expressed in senescing leaf tissue, and we were unsuccessful
at cloning any other ACO gene (using RT-PCR and degenerate prim-
ers) from this tissue. Many studies have identified at least one
member of the ACO gene family as a senescence associated gene
(SAG), for example TR-ACO3 and TR-ACO4 in white clover (Hunter
et al., 1999; Chen and McManus, 2006), LE-ACO1 in tomato (John
et al., 1995) and CP-ACO2 in papaya (Chen et al., 2003). Both MD-
ACO1 and MD-ACO2 are expressed predominantly in mature fruit
which may be associated with senescence, and MD-ACO3 is more
likely to be a photosynthetic associated gene (PAG), as it is ex-
pressed predominantly in initial bourse shoot leaf tissue. However,
MD-ACO3 is also expressed in mature green fully expanded leaf tis-
sue, and MD-ACO2 is also expressed in initial bourse shoot leaf tis-
sue and mature green fully expanded leaf tissue. Therefore MD-
ACO2 may also be a PAG.
In terms of phylogenetic analysis, it is interesting to note that
MD-ACO1, MD-ACO2 and MD-ACO3 most closely align with pear
(Pyrus pyrifolia and Pyrus communis) ACOs. MD-ACO2 shares the
closest alignment with an ACO (PP-AOX4) expressed in the imma-
ture fruit of pear, and MD-ACO1 is also associated with ACO ex-
pressed in pear fruit (JP-AOX1 and ACO1) and unripe mature pear
fruit (PC-ACO1). In contrast, MD-ACO3 shares the closest alignment
with an ACO from ripe pear fruit (JP-AOX3, PP-AOX2A and PP-
AOX2B). Unfortunately, the literature does not appear to contain
data on pear ACO expression in leaf tissue, differential expression
or even temporal expression in the fruit, but rather studies have fo-
cused on the role of ethylene, cold treatment and ACO accumula-
tion postharvest in pear fruit ripening (Lelievre et al., 1997; Gao
et al., 2002; Hiwasa et al., 2003; Fonseca et al., 2004). Broadly,
the peach ACO (PP-ACO1 and PP-ACO2) each fall into one of two
groups, with PP-ACO1 in the MD-ACO3 grouping, and PP-ACO2 in
the MD-ACO1 and MD-ACO2 grouping (Fig. 4). Interestingly PP-
ACO1 resembles MD-ACO3 as they are both expressed in young
fully expanded green leaf tissue, but PP-ACO1 also resembles MD-
ACO1 and MD-ACO2 in that they are all expressed abundantly dur-
ing the climacteric necessary for fruit ripening. However, unlike
any of the MD-ACO genes, PP-ACO1 is a SAG gene as it is expressed
abundantly in senescing leaf tissue.
In terms of protein accumulation, the antibody raised against
MD-ACO1 recognizes protein accumulated in climacteric apple,
but does not recognize ACO protein accumulated in young fruit,
initial leaf tissue or senescent leaf tissue extract (Fig. 5). These re-
sults are in agreement with Dilley et al. (1993) who report that
antibodies raised against MD-ACO1 (clone pAE12) were unable to
detect protein in the preclimacteric Golden Delicious cultivar
(nor was ACO activity detected). Further, no MD-ACO proteins
were detected during the preclimacteric stage in the Granny Smith
cultivar, using antibodies raised against either recombinant MD-
ACO1 (clone pAE12) or against MD-ACO1 purified from apple (Lara
and Vendrell, 2000). In contrast to MD-ACO1, antibodies raised
against recombinant MD-ACO3 recognize protein predominantly
in young leaf tissue, but also in mature-green leaf tissue, and in
young fruit tissue.
These protein accumulation studies confirm the gene expres-
sion studies presented. They also confirm that, again, none of the
genes under study here display expression or protein accumulation
in senescent leaf tissue. As the senescence associated genes LE-
ACO1, PP-ACO1 and CP-ACO2 are expressed both in mature fruit tis-
sue and in senescing leaf tissue, it is unusual that the MD-ACO
(transcript and protein) which increase in abundance during the
climacteric (MD-ACO1 and MD-ACO2) do not also show a senes-
cence-associated increase at the onset of senescence in the leaves
of apple.
MD-ACO1
MD-ACO2
MD-ACO3
Km
(
l
M)
89.39 9.71
15.15 0.73
6.6 ꢀ 10ꢁ2
401.03 22.1
12.94 0.97
3.44 ꢀ 10ꢁ2
0.86 ꢀ 10ꢁ4
244.5 14.2
18.94 0.69
9.14 ꢀ 10ꢁ2
3.8 ꢀ 10ꢁ4
Vmax (nmol mgꢁ1 minꢁ1
)
Kcat sꢁ1
Kcat/Km
(
l
M sꢁ1
)
7.38 ꢀ 10ꢁ4
Table 3
Thermal stability of Malus domestica ACC oxidases at the incubation temperatures, as
indicated.
Temperature
10 min
20 min
30 min
60 min
75%
25 °C
35 °C
45 °C
15%a,b
40%
85–90%
30%
70%
50%
80%
a
The data is presented as a percentage of the enzyme activity lost at each
temperature and at each time (min) of sampling and represents the mean of three
experiments.
b
Data is pooled for MD-ACO1, MD-ACO2, and MD-ACO3.
of MD-ACO3 may be present. To determine the relationship of MD-
ACO1 and MD-ACO2, and to determine the copy number of MD-
ACO3 more unequivocably, Southern analysis using a greater array
of restriction enzymes is required.
Expression studies with MD-ACO1 showed that the gene is ex-
pressed in mature fruit, with negligible amounts in young fruit,
and no transcripts were visible at all in leaf tissue. This is consis-
tent with observations from many studies on MD-ACO1 expression
during fruit ripening. For example, the pAP4 gene (MD-ACO1) from
apple fruit was found to be up-regulated during the ripening of cvs
Gala, Braeburn and Granny Smith. Expression in Gala was detected
earlier than in Braeburn and Granny Smith cultivars (Atkinson
et al., 1998), relative to the internal base-line levels of ethylene
concentration for each of the cultivars. Further, mRNA (clone
pAE12) encoding MD-ACO1 accumulated to high levels over the
course of the ethylene climacteric. For example, Ross et al.
(1992) found MD-ACO1 (clone pAP4) probes did not hybridize to
young immature apple fruit tissue extract, but as ripening pro-
gressed the degree of hybridization increased. Such an increase
in apple ACO mRNA transcripts corresponds with the climacteric
rise in ethylene biosynthesis from the mature green stage (Dilley
et al., 1995; Lelievre et al., 1997; Bleecker and Kende, 2000;
Giovannoni, 2001). Finally, fragments of the MD-ACO1 (clone
pAP4) promoter (ꢁ1966 bp upstream of ATG) fused to the
b-glucuronidase (GUS) reporter gene and transformed into tomato
were found to be active only in the fruit in a ripening specific
pattern (Atkinson et al., 1998).
MD-ACO1 in this study also appears to have a similar expression
pattern in fruit tissue to LE-ACO1 (Barry et al., 1996; Blume and
Grierson, 1997) and to the LE-ACO4, both from tomato (Nakatsuka
et al., 1998), as well as PP-ACO1 from peach (Ruperti et al., 2001;
Rasori et al., 2003; Moon and Callahan, 2004) and PA-ACO1 from
apricot (Mbéguié-A-Mbéguié et al., 1999). Both LE-ACO1 and LE-
ACO4 are expressed in immature green and mature green fruit,
with the abundance increasing greatly during the climacteric, par-
ticularly for LE-ACO1 (Nakatsuka et al., 1998). In common with MD-
ACO1, LE-ACO4 is not detectable in leaf tissue (Nakatsuka et al.,
1998), whereas LE-ACO1 transcripts have been observed to in-
crease (up to 27-fold) at the onset of leaf senescence (Barry et al.,
1996). Given that both the tomato LE-ACO1 and the peach PP-
ACO1 genes are expressed abundantly during the fruit climacteric
and during leaf senescence, the prediction that either MD-ACO1
or MD-ACO2 are good candidates for the senescence associated
gene (SAG) in apple leaf seems reasonable. However, from the

356
J.E. Binnie, M.T. McManus / Phytochemistry 70 (2009) 348–360
The lack of expression of these particular ACO genes during leaf
ferred isoform with ACC as substrate, particularly when compared
ACC
senescence may be significant as fruit removal appears to nega-
tively impact on subsequent MD-ACO3 expression and MD-ACO3
accumulation. From other studies it is well established that fruit
removal does influence leaf physiology in apple trees (Tartachnyk
and Blanke, 2004) and peach trees (Nii, 1997). For example, de-
layed apple fruit harvest has been observed to enhance autumn
leaf photosynthesis, delay chlorophyll degradation and the nitro-
gen content declined less rapidly when compared with the leaves
from the trees where the fruit had been harvested (Tartachnyk
and Blanke, 2004). Such an apparent delaying of apple leaf senes-
cence by delaying the fruit harvest, and conversely inducing the
earlier onset of senescence by removing the fruit, may underlie
the regulation of MD-ACO3, as MD-ACO3 is expressed (and MD-
ACO3 accumulated) prior to fruit removal, but were undetectable
following fruit harvest. The timing of ACO gene expression in leaf
tissue as it relates to fruit longevity has not previously been re-
ported, but does underline the classification of MD-ACO3 as a
PAG gene. It seems likely that, in agreement with many other stud-
ies, a senescence-associated MD-ACO gene does exist. However,
using the screening procedures reported her, no such gene was
identified.
This study has also investigated the kinetic properties of three
MD-ACO isoforms expressed in E. coli. ACO extracted from apple
fruit tissue has been extensively characterized biochemically
(Dong et al., 1992a,b; Fernández-Maculet and Yang, 1992; Kuai
and Dilley, 1992; Poneleit and Dilley, 1992; Pirrung et al., 1993;
Dilley et al., 1993; Dupille et al., 1993; Mizutani et al., 1995). Fur-
ther, MD-ACO1 has also been expressed both in E. coli (clone pAE12)
(Charng et al., 1997; Charng et al., 2001; Yoo et al., 2006), and in
yeast (clone pAP4) (Wilson et al., 1993). However, the current
study is the first to compare the multiple members of the apple
gene family.
The kinetic properties of the three MD-ACO isoforms do not
markedly differ in terms of co-factors and co-substrate require-
ments. However, less NaHCO3 is required for optimal enzymatic
activity for MD-ACO purified from mature apple fruit (Dong et al.,
1992a,b; Pirrung et al., 1993) when compared with the concentra-
tion required for recombinant MD-ACOs expressed in E. coli in this
study (30 mM). Similarly, although ACOs exhibit an absolute
requirement in vitro for ascorbate, the amount required for optimal
activity of the MD-ACOs purified from mature apple fruit (1–
10 mM), is again lower when compared with the amount of ascor-
bate required for recombinant MD-ACOs (20–30 mM). It is also
interesting that increased concentrations of ascorbate have been
observed to decrease the MD-ACO activities (in this study), which
is consistent with the LE-ACOs expressed in yeast (Bidonde et al.,
1998), LE-ACO1 expressed in E. coli (Zhang et al., 1995) and in the
MGI and SE1 isoforms purified from white clover leaf tissue (Gong
and McManus, 2000).
In terms of thermostability, all three recombinant isoforms ap-
pear equally unstable. However, in this study following incubation
at 25 °C for 60 min, the loss of enzyme activity decreased ꢂ75%,
whereas a purified ACO from Golden Delicious fruit displayed loss
of activity when incubated at a temperature of 23 °C, with a t½ of
ꢂ2 h (Bolitho et al., 1997). Further, an ACO purified to near homo-
geneity from pear fruit displays a t½ of 60 min at an incubation
temperature of 28 °C (Vioque and Castellano, 1998). Thus, both of
these ACOs purified from plant tissues appear to be more stable
than the recombinant MD-ACOs expressed in E. coli in this study.
Examination of kinetic constants for the three isoforms did re-
veal some significant differences, and which may also allude to
the function of these isoforms. The KmACC value for MD-ACO1 is sig-
nificantly lower than that calculated for MD-ACO2 and MD-ACO3,
although the turnover number is broadly similar. However, when
the KmACC/Kcat is compared, then MD-ACO1 emerges as the pre-
to MD-ACO2. The differences in Km
between MD-ACO1 and MD-
ACO2 may also be significant physiologically when the likely levels
of ACC in vivo are considered. For example, ACC levels have been
shown to increase from ca. 1 nmol gꢁ1 DW in the peel and the pulp
of cv. Golden Delicious prior to the climacteric to 57.32 nmol gꢁ1
DW in the peel and 47.19 nmol gꢁ1 DW in the pulp during the ap-
ple fruit climacteric (Tan and Bangerth, 2000; Lara and Vendrell,
2000), and it is the isoform with the highest affinity for ACC
(MD-ACO1) that accumulates at the developmental stage in fruit
that accumulates the higher ACC level. This suggests a mechanism
to ensure maximum autocatalytic ethylene production during the
climacteric without ACC becoming limiting. Of further interest is
that two oxidase genes (MD-ACO2, MD-ACO3) are expressed in
pre-climacteric fruit, with the protein product of at least one
(MD-ACO3) accumulating at this developmental stage. The expres-
sion of MD-ACO2 is higher than MD-ACO3 suggesting that MD-ACO2
is the prominent isoform in pre-climacteric fruit. However, the rel-
atively low affinity for ACC and the lower ACC levels at this devel-
opmental stage may provide a mechanism by which only low
levels of ethylene are produced – the system I ethylene – a phe-
nomenon commonly observed in pre-climacteric fruit. In our
expression studies, low expression of MD-ACO1 was also detected
at the pre-climacteric stage (although no protein accumulation
was detected). This low level of expression, coupled with a very
low accumulation of protein, contributes to a high likelihood that
significant ethylene production will not occur. In other studies
where the expression of a climacteric-associated ACC oxidase gene
has been examined, the expression of this gene can often be in-
duced by exogenous ethylene at the pre-climacteric stages of fruit
ripening (Tonutti et al., 1997; Whittaker et al., 1997; Liu et al.,
1999), and as such may resemble MD-ACO1 in this study (although
we have not determined whether the expression of MD-ACO1 is in-
duced by ethylene). However, where expression of different mem-
bers of the ACC oxidase have been dissected during fruit
development, a more complex pattern emerges. In tomato fruit
development, LeACO1, LeACO3 and LeACO4 are proposed to be
responsible for the system 1 (pre-climacteric) ethylene, while
LeACO1 and LeACO4 are responsible for the autocatalytic climac-
teric-associated system 2 ethylene production (Cara and Giovan-
noni, 2008). This complexity is akin to the pattern in this study
where the expression of other genes, in addition to MD-ACO1, is
apparent in young and mature apple fruit.
In leaf tissue, a burst of ethylene evolution has been observed at
bud-break in many species (Osborne, 1991), with higher levels of
ACC also reported in young leaf tissue, which then decrease as
the leaf matures (Hunter et al., 1999). However, in mature leaf tis-
sue, ethylene levels are generally lower (ca. 0.5–2.0 nL gꢁ1 FW) be-
fore increasing again during leaf senescence (Hunter et al., 1999). It
is likely, therefore, that the accumulation of MD-ACO3, with a high-
er Km (lower affinity for ACC), coupled with the lower levels of ACC
may result in a generally lower rate of ethylene production, gener-
ally indicative of mature leaves (although it should be noted that
neither ACC nor the rate of ethylene evolution has been measured
from these leaf tissues).
4. Conclusions
Two further ACO genes, designated MD-ACO2 and MD-ACO3,
have been isolated and the expression and accumulation of the
protein products has been elucidated in addition to the well char-
acterised fruit-ripening associated gene, MD-ACO1. The expression
of MD-ACO2 and MD-ACO3 also occurs in fruit tissue, with MD-
ACO3 predominantly expressed in the pre-climacteric fruit. In leaf
tissue, the expression of MD-ACO3 is again associated with younger
leaf tissues, and ceases with fruit harvest. In this study, no leaf

J.E. Binnie, M.T. McManus / Phytochemistry 70 (2009) 348–360
357
Table 4
senescence-associated ACO has been identified. Kinetic analysis of
Primer sequences used in the study.
the three enzymes expressed in E. coli, has identified differences in
biochemical properties which is postulated to provide a further tier
of control in terms of the regulation of ethylene biosynthetic dur-
ing fruit maturation and leaf development.
Oligo name
Primer sequence (50–30)
ACOF1
ECOR1
GT GAATTC GAY GCN TGY SAN AAY TGG GG
TCG TCTAGA TC RAA NCK MGG YTC YTT
E101
E102
GTGAATTC GCN TGY GAR AAY TGG GGH TT
TCG TCTAGA GYT CYT TNG CYT GRA AYT T
GAC GAC CAT ATG GCG ACT TTC CCA GTT GTT G
GAC GAC CTC GAG TCA GGC AGT TGC AAC AGG
GAC GAC CAT ATG GCA ACT TTC CCA GTT GTT G
GAC GAC CTC GAG TCA ATT TTG ATT TCT TTT TGT C
GAC GAC CAT ATG GAG AAC TTC CCA GTT ATC AAC C
GAC GAC CTC GAG TCA AGC AGT ACT TAT AAC TGG ACC
GAY TAC ATG AAG CTS TAY KCT GG
PX-MD1F
PX-MD1R
PX-MD2F
PX-MD2R
PX-MD3F
PX-MD3R
PBMF
5. Experimental
5.1. Plant material
Leaves and fruit at the appropriate developmental stage were
harvested from randomly pre-tagged bourse shoots, at the appro-
priate time of day (11.00 am) and time of year, from fifteen Malus
domestica Borkh. cv. Royal Gala (10 year old) trees which had been
grafted onto M9 dwarfing rootstock and grown at latitude 30° 400;
longitude 176° 530 at the Hawkes Bay Research Centre of HortRe-
search, Havelock North, New Zealand. Harvested leaf tissue was
frozen in liquid nitrogen immediately, and stored at ꢁ80 °C until
use. Total chlorophyll content was used to determine leaf develop-
ment. Young leaf tissue was designated the developmental stage
when chlorophyll content was increasing (typically October–
November), the mature-green stage occurred when the chlorophyll
levels had stabilised as a maxima (typically December–March),
while senescent leaves were harvested when the chlorophyll levels
were declining (typically April–June). Fruit tissues were harvested
as young fruit (mean diameter 2 cm; 7 WAFB) and mature (at rip-
ening; 17 WAFB). Mature fruit were picked at ripening as deter-
mined by the detection of maximum ethylene evolution (the
climacteric).
PBM2F
GGA ACC CGA CAA AAA G
SQ-MD1F
SQ-MD2F
SQ-MD3F
UTR-MD1R
UTR-MD2R
UTR-MD3R
ActinF
GAA GAG TAC AGG AAG ACC ATG AAG
GAA GAT TAC AGG AAG ACC ATG AAG
GAT GAG TAC AGG AAT GTG ATG AAG
TTG TTG AGC AAA CAT TTG
CTA ATA ATT TTT ATT TTA TAT AGA TAG ATT TCA AC
CAC ATT GGT TAC TTT CTA CAA CG
GCG AAT TCT TCA CCA CYA CHG CYG ARC G
GCG GAT CCC CRA TCC ARA CAC TGT AYT TTC C
ActinR
instruction and plasmids with ACO inserts were sequenced with an
ABI Prism DNA sequencer using standard procedures. Alignment of
the nucleotide sequences were analyzed using MT Navigator
1.0.2b3 (Applied Biosystems Inc.), SequencherTM 4.2 (Genes Codes
Corp., Ann Arbor, MI, USA) and Gene Computer Group (GCG version
11.0) software programmes. Interrogation of both the National
Centre for Biotechnology Information (NCBI) nucleotide compari-
son using the Basic Logical Alignment Search Tool (BLAST) of the
Genbank database, and searches of the Expressed Sequence Tag
(EST) library for apple at HortResearch were undertaken. Full-
length cDNAs complementary to each gene were then obtained
from HortResearch with the GenBank accession numbers of
X61390 (MD-ACO1), EB14037 (MD-ACO2) and EB151006 (MD-
ACO3).
5.2. RNA Isolation
Total RNA was isolated using a hot borate method adapted by
Hunter and Reid (2001). Briefly, leaf tissue was ground to a fine
powder in liquid nitrogen and the powder extracted in a hot
(80 °C) borate buffer (0.2 M sodium borate, pH 9.0, containing
30 mM EDTA, 1% (w/v) SDS, 1% (w/v) sodium deoxycholate,
10 mM DTT, 2% (w/v) polyvinylpyrollidone (PVP-40) and 1% (v/v)
octylphenylpolyethylene glycol (IGEPAL)). After vortexing for
30 s, proteinase K was added and the mixture incubated, at 42 °C,
with shaking, for 1.0 h. A volume (0.08) of 2 M KCl was then added
and the extraction continued on ice, with shaking, for 45 min. Cel-
lular debris and denatured proteins were collected by centrifuga-
tion at 26,000g for 20 min at 4 °C, and then the supernatant was
adjusted to a final concentration of 2 M LiCl and the RNA precipi-
tated overnight at 4 °C. The RNA was collected by centrifugation
at 26,000g for 20 min at 4 °C, the pellet resuspended in sterile
water at room temperature, an equal volume of chloroform: iso-
amyl alcohol (24:1) added, the aqueous and organic phases mixed
by vortexing and then separated by centrifugation at 20,800g for
5 min at room temperature. The upper aqueous phase was col-
lected, the RNA precipitated with ethanol and then resuspended
in a small volume of water, until required.
5.4. Southern Analysis
Genomic DNA was extracted from young expanding apple
leaves using a modified method of Junghans and Metziaff (1990)
and Keb-Llanes et al. (2002). Aliquots containing 25
lg of DNA
were digested to completion with EcoRI, HindIII and XbaI (Roche),
and fractionated on a 0.8% (w/v) agarose gel. The DNA was trans-
ferred to a HybondTM-N+ membrane (Amersham) by downward
capillary transfer in 10 x SSC at pH 7, and probed with cDNA frag-
ments from either 32P-labelled MD-ACO1, 32P-labelled MD-ACO2 or
32P-labelled MD-ACO3. To generate gene-specific probes, the fol-
lowing primer sets were used: PBMF/UTR-MD1R (MD-ACO1),
PBM2F/UTR-MD2R (MD-ACO2) and PBMF/UTR-MD3R (MD-ACO3)
(Table 4). Membrane hybridization and washing was carried out
using the method of Church and Gilbert (1984), with hybridization
at 65 °C overnight, and the final stringent wash with 0.1ꢀ SSPE at
65 °C.
5.3. Isolation and identification of MD-ACOs
5.5. Expression studies using semi-quantitative (sq)RT-PCR
Total RNA (5
lg) was subjected to reverse transcriptase (RT)
treatment using an oligo d(T)15 primer, with putative ACO cDNAs
amplified by the polymerase chain reaction (PCR) using initially,
degenerate oligonucleotide primers known to bind to highly con-
served regions within ACO genes. Nested PCR primers were used
to amplify ACO sequences in two rounds of PCR using ACOF1 and
ACOR1 as first round primers and E101 and E102 as the second
round primers (Table 4). The PCR products were cloned into the
pGEMÒ-T Easy Vector system (Promega) using the manufacturer’s
Reverse transcription of RNA was carried out using total RNA
(5 lg) and random primers (10 lM) in a total volume of 10 lL,
the mixture incubated for 3 min at 70 °C, quenched on ice for
2 min and collected by a brief pulse centrifugation at 4 °C. On ice,
4
l
L of 5ꢀ Expand reverse transcriptase buffer (Roche), 2
DTT, 2 L of dNTP mix and 0.5 L of RNase inhibitor were added,
the contents mixed and pulse centrifuged briefly before incubating
at 42 °C for 2 min. After which, 1 L of Expand reverse transcrip-
lL of
l
l
l

358
J.E. Binnie, M.T. McManus / Phytochemistry 70 (2009) 348–360
tase (Roche) was added and the reaction incubated for 1 h at 42 °C.
For PCR, each reaction contained 2 L of the appropriate ACO or b-
actin primers, 4 L of a 1:4 ratio of 18S rRNA primers to competi-
mers mix, 25 L of Mastermix (Promega) in a total volume of
45 L. Typically, 5 L of the appropriate cDNA (the RT-reaction
were collected on ice and assessed for the purity of recombinant
ACO using SDS–PAGE and western analysis. Fractions of interest
were stored at ꢁ20 °C prior to use for antibody production or for
ACO assays. The protein content of extracts and column fractions,
as appropriate was determined using the the method of Bradford
(1979), and SDS–PAGE performed according to Laemmli (1970).
l
l
l
l
l
description described previously) was added and typical parame-
ters for PCR were, typically, one cycle at 94 °C for 2 min; 35 cycles
at 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min with a final
incubation step at 72 °C for 5 min. To amplify each gene specifi-
cally, and to avoid any genomic DNA contamination, exon span-
ning primers were designed as follows: SQ-MD1F/UTR-MD1R
(MD-ACO1), SQ-MD2F/UTR-MD2R (MD-ACO2) and SQ-MD3F/UTR-
MD3R (MD-ACO3) (Table 4). To visual PCR product accumulation
using radiolabelled probes, the DNA fragments generated by PCR
were transferred to HybondTM-N+ nylon membrane by the down-
ward capillary transfer method, except the gel was not depurinat-
ed, denatured or neutralised before transfer, and the transfer time
was 5 h. The membrane was probed with 32P-labelled MD-ACO1,
32P-labelled MD-ACO2 or 32P-labelled MD-ACO3 generated using
the same primer sets described for Southern analysis.
5.7. Protein extraction from apple tissue
ACO was extracted from apple leaf and fruit tissue using a pro-
cedure, based on that of Britsch and Grisebach (1986) for the
extraction of flavanone-3-hydroxylase and modified by
Fernández-Maculet and Yang (1992) for ACO extraction from the
fruits of apple. Leaf tissue (free from the midrib) or apple fruit tis-
sue was ground under liquid nitrogen to a fine powder, extraction
buffer (100 mM Tris-HCl, pH 7.5, containing 30 mM sodium-ascor-
bate, 10% (v/v) glycerol, 4 mM DTT, 1.0% (v/v) Triton X-100) was
added in a 3:1 (v/w) ratio to the ground frozen powder, and the
mixture vortexed vigorously for 2 to 3 min before centrifugation
at 20,800g for 20 min at 4 °C. The supernatant (crude extract)
was then used for further experiments.
5.6. Expression and purification of recombinant proteins
5.8. Production of primary polyclonal antibodies and western analysis
PCR products, corresponding to each of the MD-ACO genes were
generated using the following primer sets: PX-MD1F/PX-MD1R
(MD-ACO1), PX-MD2F/PX-MD2R (MD-ACO2) and PX-MD3F/PX-
MD3R (MD-ACO3) (Table 4). The PCR products were cloned into
pGEMÒ-T Easy Vector system, digested with the appropriate
restriction enzymes and directionally ligated into the correspond-
ing sites of the pProEX-1 (Gibco-BRL) expression vector system.
The resulting plasmid was introduced into E. coli (BL-21) compe-
tent cells and the cells grown in LB broth with 100 mg/mL ampicil-
lin, initially at 37 °C until the OD600 of the culture reach ca. 0.6, and
then the temperature was shifted to 20 °C for 15 min before adding
ITPG to a final concentration of 0.6 mM. The culture was then incu-
bated at 20 °C for approximately 16 h before the cells were har-
vested by centrifugation at 3000 g for 20 min at 4 °C, the
supernatant removed and the cells resuspended in Lysis Buffer A
(containing 50 mM sodium phosphate buffer, pH 8.5, containing
300 mM NaCl, 10 mM imidazole and 10% (v/v) glycerol). Following
cell disruption using a Virsonic digital 475 Ultrosomic cell dis-
rupter (The VirTis Company), the debris was removed by centrifu-
gation at 12,000 g for 20 min at 4 °C. The His-tagged recombinant
ACO proteins in the supernatant were purified by affinity chroma-
tography using nickel nitrilotriacetic acid resin (Ni-NTA). To do
this, the column was first pre-equilibrated with 8–10 volumes of
Buffer A, the supernatant gently loaded onto the column, the col-
umn washed with ten volumes of Buffer A, two volumes of Buffer
B (50 mM sodium phosphate buffer, pH 8.5, containing 300 mM
NaCl, 20 mM imidazole and 10% (v/v) glycerol) and the His-tagged
ACO fusion protein then eluted with ten volumes of Buffer C
(50 mM sodium phosphate buffer, pH 8.5, containing 300 mM
NaCl, 250 mM imidazole and 10% (v/v) glycerol). The eluate frac-
tions containing proteins of interest were the pooled and ex-
changed into FPLC Buffer A (25 mM Tris-HCl, pH 7.5, containing
Polyclonal antibodies were raised, in New Zealand white rab-
bits, against affinity and anion exchange column chromatography
His-tagged recombinant MD-ACO proteins. The primary immunisa-
tion comprised 400
Freund’s adjuvant, followed by three boosts, at 4–5 week intervals,
with 300 g of fusion protein emulsified with Freund’s incomplete
lg of fusion protein emulsified with complete
l
adjuvant. For western analysis, proteins separated by SDS–PAGE
were electrophoretically transferred to polyvinylidine difluoride
membrane (PVDF; Perkin ElmerTM, PolyscreenÒ) using a Trans-Blot
Electrophoretic Transfer Cell (Bio-Rad) in 25 mM Tris, 190 mM gly-
cine and 10% (v/v) methanol, at pH 8.3. Antibody incubation and
development of antibody recognition was carried out using stan-
dard procedures (Hunter et al., 1999).
5.9. ACC oxidase assays
ACO activity was measured essentially according to the method
described by Ververidis and John (1991). The standard reaction
mixture comprised 50 mM phosphate buffer, pH 7.5), containing
10% (v/v) glycerol, 1 mM ACC, 2 mM DTT, 30 mM sodium ascor-
bate, 20 lM FeSO4 and 30 mM NaHCO3. To start the reaction,
0.2 mL enzyme samples were pipetted into 4.5 mL capacity vacu-
tainer tubes (Becton Dickinson) containing the reaction mixture
(0.8 mL), pre-equilibrated at 30 °C, and the reactions incubated,
with shaking, at 180 rpm for 20 min at 30 °C. After this time,
1 mL gas samples were removed, and the ethylene content deter-
mined using a Model GC-8A gas chromatograph (Shimadzu Corp.,
Kyoto, Japan) fitted with a flame ionization detector. For ethylene
analysis, a 2.5 ꢀ 3 mm I.D. glass column prepacked with Porapak-
Q with a mesh size of 80/100 (Alltech Associates Inc., Deerfield,
Il., USA) was used, with nitrogen as the carrier gas with a flow rate
of 50 mL minꢁ1 and the injector/detector temperatures were set at
85 and 150 °C, respectively. To determine pH optima, a range of pH
values were constructed using a standard 100 mM triphosphate
activity buffer adjusted to the appropriate pH with phosphoric
acid. ACO assays were then performed as described previously.
The optimal requirements for co-substrate and cofactors were
determined at the optimal pH for each ACO assessed, and at satu-
rating ACC concentrations. The apparent Km and Vmax values of
each of the ACO isoforms was determined by triplicate measure-
ments of initial velocity at different ACC concentrations, at the
optimal pH for each isoform, with rate constants calculated using
2 mM DTT, 15 mM sodium ascorbate and 10 lM PA using Sepha-
dexÒ G-25 resin (Pharmacia Biotech). In order to retain ACO activ-
ity, all steps necessary for the purification of the recombinant
proteins were carried out within the same day and the extract
stored at ꢁ20 °C overnight for further purification the next day.
For FPLC, a Mono-Q prepacked HR 5/5 strong anion column (Amer-
sham/Pharmacia Biotech) was equilibrated with at least 5 volumes
of buffer A before sample loading and proteins were eluted within
an linear salt gradient of 100% buffer A: 0% buffer B (25 mM Tris-
HCl, pH 7.5, containing 2 mM DTT, 15 mM sodium ascorbate,
10 lM PA and 1.0 M NaCl) to 0% buffer A: 100% buffer B. Fractions

J.E. Binnie, M.T. McManus / Phytochemistry 70 (2009) 348–360
359
Chen, C.M., McManus, M.T., 2006. Expression of 1-aminocyclopropane-1-
carboxylate (ACC) oxidase genes during the development of vegetative tissues
in white clover (Trifolium repens L.) is regulated by ontological cues. Plant
Molecular Biology 60, 451–467.
Chen, Y.T., Lee, Y.R., Yang, C.Y., Wang, Y.T., Yang, S.F., Shaw, J.F., 2003. A novel
papaya ACC oxidase gene (CP-ACO2) associated with late stage fruit ripening
and leaf senescence. Plant Science 164, 531–540.
Church, G.M., Gilbert, W., 1984. Genomic sequencing. Proceedings of the National
Academy of Sciences, USA 81, 1991–1995.
Cin, V.D., Danesin, M., Boschetti, A., Dorigoni, A., Ramina, A., 2005. Ethylene
biosynthesis and perception in apple fruitlet abscission (Malus domestica L.
Borck). Journal of Experimental Botany 56, 2995–3005.
ENZYPLOT (Walker,1997) and ENZFITTER version 2.0 (BioSoft,
Cambridge, UK). The Kcat was determined from the equation: prod-
uct (mol sꢁ1)/enzyme (mol). To assess the thermal stability of each
isoform, aliquots of each recombinant protein were incubated at
either 25, 35 or 45 °C, as appropriate and aliquots removed at 10,
20, 30, 60, 90, 120 and 180 min intervals and ACO activity deter-
mined, as described.
Acknowledgements
Costa, F., Stella, S., van de Weg, W.E., Guerra, W., Cecchinel, M., Dallavia, J., Koller, B.,
Sansavini, S., 2005. Role of the genes Md-ACO1 and Md-ACS1 in ethylene
production and shelf life of apple (Malus domestica Borkh). Euphytica 141, 181–
190.
Dilley, D.R., Wang, Z., Kadyrzhanova, D.K., 2003. Structure-function analysis of ACC
oxidase by site-directed mutagenesis. In: Vendrell, M., Klee, H., Pech, J.C.,
Romojaro, F. (Eds.), Biology and Biotechnology of the Plant Hormone Ethylene
III. IOA Press, The Netherlands, pp. 9–14.
Dilley, D.R., Kuai, J., Wilson, I.D., Pekker, Y., Zhu, Y., Burmeister, D.M., Beaudry, R.M.,
1995. Molecular biology investigation of ACC oxidase and its expression
attending apple fruit ripening. Acta Horticulturae 379, 25–39.
Dilley, D.R., Kuai, J., Poneleit, L., Zhu, Y., Pekker, Y., Wilson, ID., Burmeister, D.M.,
Gran, C., Bowers, A., 1993. Purification and characterization of ACC oxidase and
its expression during ripening in apple fruit. In: Pech, J.C., Latché, A., Balagué, C.
(Eds.), Cellular and Molecular Aspects of the Plant Hormone Ethylene. Kluwer
Academic Publishers, Dordreecht, The Netherlands, pp. 6–52.
We thank Dr. Robert Simpson and Dr. Andrew Allan, The Horti-
cultural and Food Research Institute of New Zealand (HortRe-
search) for assistance with the EST mining and sequencing, and
Drs Stuart Tustin and Jens Wunsche (HortResearch), for advice dur-
ing the experimental period of this study, and Professor Peter Lock-
hart (IMBS, Massey University) for assistance with the
phylogenetic analysis. The study was supported by a New Zealand
Science Foundation Bright Futures Scholarship to JEB, in associa-
tion with HortResearch.
Appendix A. Supplementary data
Dong, J.G., Fernandez-Maculet, J.C., Yang, S.F., 1992a. Purification and
characterization of 1-aminocyclopropane-1-carboxylate oxidase from apple
fruit. Proceedings of the National Academy of Sciences, USA 89, 9789–9793.
Dong, J.G., Olson, D., Silverstone, A., Yang, S.F., 1992b. Sequence of a cDNA coding for
a 1-aminocyclopropane-1-carboxylate oxidase homolog from apple fruit. Plant
Physiology 98, 1530–1531.
Dupille, E., Rombaldi, C., Lelièvre, J.M., Cleyet-Marcel, J.C., Pech, J.C., Latché, A., 1993.
Purification, properties and partial amino-acid sequence of 1-
aminocyclopropane-1-carboxylic acid oxidase from apple fruits. Planta 190,
65–70.
Fernández-Maculet, J.C., Yang, S.F., 1992. Extraction and partial characterization of
the ethylene-forming enzyme from apple fruit. Plant Physiology 99, 751–754.
Fernández-Maculet, J.C., Dong, J.G., Yang, S.F., 1993. Activation of 1-
aminocyclopropane-1-carboxylate oxidase by carbon dioxide. Biochemical
and Biophysical Research Communications 193, 1168–1173.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.phytochem.2009.01.002.
References
Abeles, F.B., Morgan, P.W., Saltveit, M.E., 1992. Ethylene in Plant Biology. In: Abeles,
F.B., Morgan, P.W., Saltveit, M.E. (Eds.), second ed. Academic Press, San Diego,
California.
Adams, D.O., Yang, S.F., 1979. Ethylene biosynthesis: identification of 1-
aminocyclopropane-1-carboxylic acid as an intermediate in the conversion of
methionine to ethylene. Proceedings of the National Academy of Sciences, USA
76, 170–174.
Atkinson, R.G., Bolitho, K.M., Wright, M.A., Iturriagagoitia-Bueno, T., Reid, S.J., Ross,
G.S., 1998. Apple ACC-oxidase and polygalacturonase: ripening-specific gene
expression and promoter analysis in transgenic tomato. Plant Molecular Biology
38, 449–460.
Barry, C.S., Blume, B., Bouzayen, M., Cooper, W., Hamilton, A.J., Grierson, D., 1996.
Differential expression of the 1-aminocyclopropane-1-carboxylate oxidase gene
family of tomato. The Plant Journal 9, 525–535.
Bidonde, S., Ferrer, M.A., Zegzouti, H., Ramassamy, S., Latché, A., Pech, J.C., Hamilton,
A.J., Grierson, D., Bouzayen, M., 1998. Expression and characterization of three
tomato 1-aminocyclopropane-1-carboxylate oxidase cDNAs in yeast. European
Journal of Biochemistry 253, 20–26.
Bleecker, A.B., Kende, H., 2000. Ethylene: a gaseous signal molecule in plants.
Annual Review of Cell and Developmental Biology 16, 1–18.
Blume, B., Grierson, D., 1997. Expression of ACC oxidase promoter-GUS fusions in
tomato and Nicotiana plumbaginifolia regulated by developmental stimuli. The
Plant Journal 12, 731–746.
Fonseca, S., Barreiro, M.G., Monteira, L., Pais, M., 2004. Cold storage induces pear
fruit ACC oxidase: enzyme activity, gene expression, promoter isolation and
analysis. In: Maturacão e Pós-Colheita, Futos e Horticolas, M. Graca Barreiro/
EAN-INIAP (Eds.), Oeiras, Portugal, pp. 151–156.
Gao, M., Matsuta, N., Nishimura, K., Tao, R., Murayama, H., Toyomasu, T., Mitsuhashi,
W., Dandekar, A.M., Hammerschlag, F.A., Saxena, P., Hicklenton, P., Bowen, P.,
Pritchard, M., Chong, C., 2002. Transformation of pear (Pyrus communis cv. ‘La
France’) with genes involved in ethylene biosynthesis. Acta Horticulturae 625,
387–393.
Giovannoni, J., 2001. Molecular biology of fruit maturation and ripening. Annual
Review of Plant Physiology and Plant Molecular Biology 52, 725–749.
Gong, D., McManus, M.T., 2000. Purification and characterization of two ACC
oxidases expressed differentially during leaf ontogeny in white clover.
Physiologia Plantarum 110, 13–21.
Hiwasa, K., Kinugasa, Y., Amano, S., Hashimoto, A., Nakano, R., Inaba, A., Kubo, Y.,
2003. Ethylene is required for both the initiation and progression of softening in
pear (Pyrus communis L.) fruit. Journal of Experimental Botany 54, 771–779.
Hunter, D.A., Reid, M.S., 2001. A simple and rapid method for isolating high quality
RNA from flower petals. Acta Horticulturae, 33–37.
Bolitho, K.M., Lay-Yee, M., Knighton, M.L., Ross, G.S., 1997. Antisense apple ACC-
oxidase RNA reduces ethylene production in transgenic tomato fruit. Plant
Science 122, 91–99.
Bouzayen, M., Felix, G., Latché, A., Pech, J.C., Boller, T., 1991. Iron: an essential
cofactor for the conversion of 1-aminocyclopropane-1-carboxylic acid to
ethylene. Planta (Heidelberg) 184, 244–247.
Hunter, D.A., Yoo, S.D., Butcher, S.M., McManus, M.T., 1999. Expression of 1-
aminocyclopropane-1-carboxylate oxidase during leaf ontogeny in white
clover. Plant Physiology 120, 131–142.
Bouzayen, M., Cooper, W., Barry, C., Zegzouti, H., Hamilton, A.J., Grierson, D., 1993.
EFE multigene family in tomato plants; expression and characterization. In:
Pech, J.C., Latché, A., Balagué, C. (Eds.), Cellular and Molecular Aspects of the
Plant Hormone Ethylene. Kluwer Academic Publishers, Dordrecht, The
Netherlands, pp. 76–81.
John, I., Drake, R., Farrell, A., Cooper, W., Lee, P., Horton, P., Grierson, D., 1995.
Delayed leaf senescence in ethylene-deficient ACC-oxidase antisense tomato
plants: molecular and physiological analysis. The Plant Journal 7, 483–490.
Junghans, H., Metziaff, M., 1990. A simple and rapid method for the preparation of
total plant DNA. BioTechniques 8, 176.
Kadyrzhanova, D.K., McCully, T.J., Warner, T., Vlachonasios, K.E., Wang, Z., Dilley,
D.R., 1999. Analysis of ACC oxidase activity by site-directed mutagenesis of
conserved amino acid residues. In: Kanellis, A.K., Chang, C., Klee, H., Bleecker,
A.B., Pech, J.C., Grierson, D. (Eds.), Biology and Biotechnology of the Plant
Hormone Ethylene II. Kluwer Academic Publishers., Dordrecht, The
Netherlands, pp. 7–12.
Kadyrzhanova, D.K., McCully, T.J., Jaworski, S.A., Ververidis, P., Vlachonasios, K.E.,
Murakami, K.G., Dilley, D.R., 1997. Structure-function analysis of ACC oxidase by
site-directed mutagenesis. In: Kanellis, A.K., Chang, C., Kende, H., Grierson, D.
(Eds.), Biology and Biotechnology of the Plant Hormone Ethylene. Kluwer
Academic Publishers., Dordrecht, The Netherlands, pp. 5–13.
Bradford, M.M., 1979.
A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye binding.
Analytical Biochemistry 72, 248–254.
Britsch, L., Grisebach, H., 1986. Purification and characterization of (2S)-flavanone
3-hydroxylase from Petunia hybrida. European Journal of Biochemistry 156,
569–577.
Cara, B., Giovannoni, J., 2008. Molecular biology of ethylene during tomato fruit
development and maturation. Plant Science 175, 106–113.
Charng, Y., Liu, Y., Dong, J.G., Yang, S.F., 1997. On 1-aminocyclopropane-1-carboxylic
acid (ACC) oxidase: degradation of
a-aminoisobutyric acid and structure-
function studies on the CO2 binding site. In: Kanellis, A.K., Chang, C., Kende, H.,
Grierson, D. (Eds.), Biology and Biotechnology of the Plant Hormone Ethylene.
Kluwer Academic Publishers, Dordrecht, The Netherlands, pp. 23–29.
Charng, Y., Chou, S.J., Jiaang, W.T., Chen, S.T., Yang, S.F., 2001. The catalytic
mechanism of 1-aminocyclopropane-1-carboxylic acid oxidase. Archives of
Biochemistry and Biophysics 385, 179–185.
Kende, H., 1993. Ethylene biosynthesis. Annual Review of Plant Physiology and
Plant Molecular Biology 44, 283–307.
Keb-Llanes, M., Gonzaliz, G., Chi-Manzanero, B., Infante, D., 2002. A rapid and simple
method for small-scale DNA extraction in Agavaceae and other tropical plants.
Plant Molecular Biology Reporter 20. 299a–299e.

360
J.E. Binnie, M.T. McManus / Phytochemistry 70 (2009) 348–360
Kuai, J., Dilley, D.R., 1992. Extraction, partial purification and characterization of 1-
aminocyclopropane-1-carboxylic acid oxidase from apple fruit. Postharvest
Biology and Technology 1, 203–211.
Poneleit, L.S., Dilley, D.R., 1993. Carbon dioxide activation of 1-aminocyclopropane-
1-carboxylate (ACC) oxidase in ethylene synthesis. Postharvest and Biology and
Technology 3, 191–199.
Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the
head of bacteriophage T4. Nature 227, 680–685.
Lara, I., Vendrell, 2000. Changes in abscisic acid levels, ethylene biosynthesis, and
protein patterns during fruit maturation of ‘Granny Smith’ apples. Journal of the
American Society of Horticultural Science 125, 183–189.
Rasori, A., Bertolasi, B., Furini, A., Bonghi, C., Tonutti, P., Ramina, A., 2003. Functional
analysis of peach ACC oxidase promoters in transgenic tomato and in ripening
peach fruit. Plant Science 165, 523–530.
Ross, G.S., Knighton, M.L., Lay-Yee, M., 1992. An ethylene-related cDNA from
ripening apples. Plant Molecular Biology 19, 231–238.
Lasserre, E., Bouquin, T., Hernandez, J.A., Bull, J., Pech, J.C., Balague, C., 1996.
Structure and expression of three genes encoding ACC oxidase homologs
from melon (Cucumis melo L.). Molecular and General Genetics 251, 81–
90.
Lasserre, E., Godard, F., Bouquin, T., Hernandez, J.A., Pech, J.C., Roby, D., Balagué,
1997. Differential activation of two ACC oxidase gene promoters from melon
during plant development and in response to pathogen attack. Molecular and
General Genetics 256, 211–222.
Lelievre, J.M., Latché, A., Jones, B., Bouzayen, M., Pech, J.C., 1997. Ethylene and fruit
ripening. Physiologia Plantarum 101, 727–739.
Liu, X., Shiomi, S., Nakatsuka, A., Kubo, Y., Nakamura, R., Inaba, A., 1999.
Characterization of ethylene biosynthesis associated with ripening in banana
fruit. Plant Physiology 121, 1257–1265.
Maliepaard, G., Alston, F.H., van Arkel, G., Brown, L.M., Chevreau, E., Dunemann, F.,
Evans, K.M., Gardiner, S., Guilford, P., van Heusden, A.W., Janse, J., Laurens, F.,
Lynn, J.R., Manganaris, A.G., den Nijs, A.P.M., Periam, N., Rikkerink, E., Roche, P.,
Ryder, C., Sansavini, S., Schmidt, H., Taretarini, S., Verhaegh, J.J., Vrielink-van
Ginkel, M., King, G.J., 1998. Aligning male and female linkage maps of apple
(Malus pumila Mill) using multi-allelic markers. Theoretical and Applied
Genetics 97, 60–73.
Ruperti, B., Bonghi, C., Rasori, A., Ramina, A., Tonutti, P., 2001. Characterization and
expression of two members of the peach 1-aminocyclopropane-1-carboxylate
oxidase gene family. Physiologia Plantarum 111, 336–344.
Shaw, J.F., Chou, Y.S., Chang, R.C., Yang, S.Y., 1996. Characterisation of the ferrous
iron binding sites of apple 1-aminocyclopropane-1-carboxylate oxidase by site-
directed mutagenesis. Biochemical and Biophysical Research Communications
225, 697–700.
Seo, Y.S., Yoo, A., Jung, J., Sung, S.K., Yang, D.R., Kim, W.T., Fee, W., 2004. The active
site and substrate-binding model of 1-aminocyclopropane-1-carboxylate
oxidase determined by site-directed mutagenesis and comparative modeling
studies. Biochemical Journal 380, 339–346.
Tan, T., Bangerth, F., 2000. Regulation of ethylene, ACC, MACC production, and ACC-
oxidase activity at various stages of maturity of apple fruit and the effect of
exogenous ethylene treatment. Gartenbauwissenschaft 65, S121–S128.
Tartachnyk, I., Blanke, M., 2004. Key processes in leaf senescence of fruiting apple
trees. Acta Horticulturae 636, 59–67.
Tonutti, P., Bonghi, C., Ruperti, B., Tornielli, G.B., Ramina, A., 1997. Ethylene
evolution and 1-aminocyclopropane-1-carboxylate oxidase gene expression
during early development and ripening of peach fruit. Journal of the American
Society of Horticultural Science 12, 642–647.
Mbéguié-A-Mbéguié, D., Chahine, H., Gomez, R.M., Gouble, B., Reich, M., Audergon,
J.M., Souty, M., Albagnac, G., Fils Lycaon, B., 1999. Molecular cloning and
expression of cDNA encoding 1-aminocyclopropane-1-carboxylate (ACC)
oxidase from apricot fruit (Prunus armeniaca). Physiologia Plantarum 105,
294–303.
Mizutani, F., Dong, J.G., Yang, S.F., 1995. Effect of pH on CO2-activated 1-
aminocyclopropane-1-carboxylate oxidase activity from apple fruit.
Phytochemistry 39, 751–755.
Ververidis, P., John, P., 1991. Complete recovery in vitro of ethylene-forming
enzyme activity. Phytochemistry 30, 725–727.
Vioque, B., Castellano, J.M., 1998. In vivo and in vitro 1-aminocyclopropane-1-
carboxylic acid oxidase activity in pear fruit: role of ascorbate and inactivation
during catalysis. Journal of Agriculture and Food Science 46, 1706–1711.
Whittaker, D.J., Smith, G.S., Gardner, R.C., 1997. Expression of ethylene biosynthetic
genes in Actinidia chinensis fruit. Plant Molecular Biology 34, 45–55.
Wiersma, P.A., Zhang, H., Lu, C., Quail, A., Toivonen, P.M.A., 2007. Survey of the
expression of genes for ethylene perception during maturation and ripening of
‘‘Sunrise” and ‘‘Golden Delicious” apple fruit. Postharvest Biology and
Biotechnology 44, 204–211.
Moon, H., Callahan, A.M., 2004. Developmental regulation of peach ACC oxidase
promoter-GUS fusions in transgenic tomato fruits. Journal of Experimental
Botany 55, 1519–1528.
Nakatsuka, A., Murachi, S., Okunishi, H., Shiomi, S., Nakano, R., Kubo, Y., Inaba, A.,
1998. Differential expression and internal feedback regulation of 1-
Wilson, I.D., Zhu, Y., Burmeister, D.M., Dilley, D.R., 1993. Apple ripening related
cDNA pAP4 confers ethylene-forming ability in transformed Saccharomyces
cerevisiae. Plant Physiology 102, 783–788.
aminocyclopropane-1-carboxylate
synthase,
1-aminocyclopropane-1-
carboxylate oxidase, and ethylene receptor genes in tomato fruit during
development and ripening. Plant Physiology 118, 1295–1305.
Nii, N., 1997. Changes of starch and sorbitol in leaves before and after removal of
fruits from peach trees. Annals of Botany 79, 139–144.
Osborne, D.J., 1991. Ethylene in leaf ontogeny and abscission. In: Mattoo, A.K.,
Suttle, J.C. (Eds.), The Plant Hormone Ethylene. CRC Press, Boca Raton, Florida,
USA, pp. 93–214.
Park, S., Sugimoto, N., Larson, M.D., Beaudry, R., Nocker van, S., 2006. Identification
of genes with potential roles in apple fruit development and biochemistry
through large-scale statistical analysis of expressed sequence tags. Plant
Physiology 141, 811–824.
Pirrung, M.C., Kaiser, L.M., Chen, J., 1993. Purification and properties of the apple
fruit ethylene-forming enzyme. Biochemistry 32, 7445–7450.
Yang, S.F., Hoffman, N.E., 1984. Ethylene biosynthesis and its regulation in higher
plants. Annual Review in Plant Physiology 35, 155–189.
Yoo, A., Seo, Y.S., Jung, J.W., Sung, S.K., Kim, W.T., Lee, W., Yang, D.R., 2006. Lys296
and Arg299 residues in the C-terminus of MD-ACO1 are essential for a 1-
aminocyclopropane-1-carboxylate oxidase enzyme activity. Journal of
Structural Biology 156, 407–420.
Zhang, Z.H., Schofield, C.J., Baldwin, J.E., Thomas, P., John, P., 1995. Expression,
purification and characterization of 1-aminocyclopropane-1-carboxylate
oxidase from tomato in Escherichia coli. Biochemical Journal 307, 77–85.
Zhu, J., Gardiner, S.E., Lay-Yee, M., 1995. Physical mapping of three fruit ripening
genes: endopolygalacturonase, ACC oxidase and ACC synthase from apple
(Malus x domestica) in an apple rootstock A106 (Malus sieboldii). Cell Research 5,
243–253.