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Plant Physiol. (1999) 120: 131-142
Expression of 1-Aminocyclopropane-1-Carboxylate
Oxidase
during Leaf Ontogeny in White Clover1
Donald A. Hunter2,
Sang Dong Yoo2,
Stephen
M. Butcher, and
Michael T. McManus*
Institute of Molecular BioSciences, Massey University, Private Bag
11222, Palmerston North, New Zealand
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ABSTRACT |
We
examined the expression of three distinct
1-aminocyclopropane-1-carboxylic acid oxidase genes during leaf
ontogeny in white clover (Trifolium repens). Significant
production of ethylene occurs at the apex, in newly initiated leaves,
and in senescent leaf tissue. We used a combination of reverse
transcriptase-polymerase chain reaction and 3 -rapid amplification of
cDNA ends to identify three distinct DNA sequences designated TRACO1,
TRACO2, and TRACO3, each with homology to
1-aminocyclopropane-1-carboxylic acid oxidase. Southern analysis
confirmed that these sequences represent three distinct genes. Northern
analysis revealed that TRACO1 is expressed specifically in the apex and
TRACO2 is expressed in the apex and in developing and mature green
leaves, with maximum expression in developing leaf tissue. The third
gene, TRACO3, is expressed in senescent leaf tissue. Antibodies were
raised to each gene product expressed in Escherichia
coli, and western analysis showed that the TRACO1
antibody recognizes a protein of approximately 205 kD (as determined by
gradient sodium dodecyl sulfate-polyacylamide gel electrophoresis) that
is expressed preferentially in apical tissue. The TRACO2 antibody
recognizes a protein of approximately 36.4 kD (as determined by
gradient sodium dodecyl sulfate-polyacylamide gel electrophoresis) that
is expressed in the apex and in developing and mature green leaves,
with maximum expression in mature green tissue. No protein recognition
by the TRACO3 antibody could be detected in senescent tissue or at any
other stage of leaf development.
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INTRODUCTION |
The plant hormone ethylene is an important regulator of several
physiological processes in higher plants (Abeles et al., 1992 ) and also
functions as a mediator of responses to external stimuli, such as
wounding, flooding, and pathogen invasion (Kende, 1993). The
biosynthetic pathway of the hormone in higher plants has now been
characterized (Adams and Yang, 1979) and two committed enzymes in the
pathway, ACC synthase (EC 4.4.1.14) and ACC oxidase (EC 1.4.3), have
been identified (Yang and Hoffman, 1984; Kende, 1993).
ACC synthase is recognized as the rate-determining step in the
ethylene-biosynthesis pathway, and many inducers are proposed to act by
stimulation of this enzyme (Yang and Hoffman, 1984; Theologis, 1992;
Kende, 1993). The enzyme is known to be coded for by a multigene family
in several plant species, with many of these genes cloned from a wide
variety of tissues and in response to a variety of stimuli (Fluhr and
Mattoo, 1996).
In contrast, the ability of most plant tissues to convert ACC to
ethylene was interpreted originally as evidence that the regulation of
ACC oxidase is not a major control point of ethylene biosynthesis (Yang
and Hoffman, 1984). More recently, two significant advances have
provided the foundation for more detailed biochemical analysis of the
enzyme. The first was the expression of a cDNA, designated pTOM13,
coding for the putative ethylene-forming enzyme (Hamilton et al., 1990)
in yeast (Hamilton et al., 1991), and another highly homologous
cDNA, pPHTOM5, in Xenopus laevis oocytes (Spanu et al.,
1991). In both of these studies the transformants could convert ACC to
ethylene and the trans isomer of the ACC analog
1-amino-2-ethylcyclopropane-1-carboxylic acid to ethylene in preference
to the cis isomer.
The second significant advance was the demonstration that, because the
amino acid sequence of pTOM 13 is similar to the enzyme flavanone
3-hydroxylase, complete recovery of enzyme activity in vitro from melon
fruit could be achieved using factors shown to preserve the activity of
the hydroxylase (Ververidis and John, 1991). ACC oxidase has now been
purified to homogeneity and characterized from apple fruits (Dong et
al., 1992; Dupille et al., 1993; Pirrung et al., 1993) and partially
purified and characterized from a range of tissues, including apple
(Fernandez-Maculet and Yang, 1992; Kuai and Dilley, 1992), avocado
(McGarvey and Christoffersen, 1992), pear (Vioque and Castellano,
1994), and citrus (Dupille and Zacarias, 1996).
In concert with these biochemical studies, genes coding for ethylene
have now been cloned from a wide variety of tissues in many plants
(Fluhr and Mattoo, 1996). Of particular significance is the view that
the expression of the ACC oxidase gene family, like that of ACC
synthase genes, is highly regulated in plants and constitutes an extra
tier of control of ethylene biosynthesis. Differential expression of
ACC oxidase genes has been observed in orchid flowers (Nadeau et al.,
1993), mung bean epicotyls (Kim and Yang, 1994), petunia floral tissues
(Tang et al., 1994; Tang and Woodson, 1996), broccoli floral tissue
(Pogson et al., 1995), tomato (Barry et al., 1996) and melon leaf
tissues (Lasserre et al., 1996, 1997), carnation floral tissues (ten
Have and Woltering, 1997), sunflower seedling tissue (Liu et al.,
1997), geranium floral tissue (Clark et al., 1997), and leaf tissue of
Nicotiana glutinosa (Kim et al., 1998). However, compared
with fruit and floral tissue, fewer studies have been undertaken of the
regulation of ACC oxidase gene expression during leaf development (John
et al., 1995; Barry et al., 1996; Lasserre et al., 1996; Bouquin et
al., 1997; Kim et al., 1998), even though ethylene is considered an
important regulator of leaf ontogeny in higher plants (Osborne, 1991).
In this study we used the stoloniferous growth habit of white clover
(Trifolium repens) as a system to provide leaf tissue at all
developmental stages in a perfectly replicated fashion. Three distinct
DNA sequences with homology to ACC oxidase were generated using RT-PCR
and 3 RACE, and Southern analysis confirmed that these sequences are
complementary to distinct genes. We show here that these three genes
are differentially expressed during leaf ontogeny in white clover. We
also report the expression of these genes with respect to ethylene
evolution, ACC oxidase enzyme activity, and protein accumulation and
then interpret the results as further evidence for the highly
coordinated regulation of ACC oxidase expression in plants.
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MATERIALS AND METHODS |
Plant Material and Tissue Sampling
Stock plants of white clover (Trifolium repens L. genotype 10F; AgResearch Grasslands, New Zealand) were grown under
natural light in 8-L planter bags in a greenhouse maintained at a
minimum temperature of 18°C (day) and 12°C (night) and vented at
25°C (day) and 18°C (night). Apical cuttings with two or three
nodes were taken from these stock plants, and all leaves were excised at the petiole/stolon junction except the youngest fully emerged leaf.
The cuttings were placed with the basal node buried in a bark/pumice
potting mix, and as the stolon grew, the apex was trained to direct
growth over a dry matrix provided by white polythene sheeting.
At weekly intervals, any outgrowths from the axillary buds were excised
to maintain a single (unbranched) stolon. Once we achieved a consistent
program of leaf development from initiation at the apex to senescence
over 16 to 20 nodes, we harvested the leaf tissue. For tissue-sampling
purposes, leaf 1 was designated as the first (unfolded) leaf protruding
clearly from its sheath, and the apex was defined as all tissue distal
to the leaf 1 node.
Determination of Leaf Chlorophyll
Chlorophyll determinations were made essentially using the method
of Moran and Porath (1980). Up to 400 mg of freshly harvested leaf
material was immersed in 5 mL of cold (4°C)
N,N-dimethylformamide, the mixture was incubated in darkness
at 4°C for 48 h, and the A664.5 and
A647 of the extractant were determined. We
calculated the chlorophyll concentrations as described by Inskeep and
Bloom (1985).
Measurements of Ethylene Evolution
Individual attached leaves ranging from nodes 2 to 16 were
enclosed in 30-mL plastic containers with a slot cut to accommodate the
petiole. Apex and leaf 1 tissues were enclosed in a 16.5-mL plastic container. The petiole (or stolon for the apex) was held in
place with petroleum jelly, the containers were sealed, and after
1 h, a 1-mL gas sample was removed and the concentration of
ethylene measured using a gas chromatograph (model 10S70+, Photovac,
Markham, Ontario, Canada). We used the method of Lizada and Yang (1979)
to measure the ACC content.
Nucleic Acid Isolations
Genomic DNA was isolated from leaf tissue using a method modified
from that of Junghans and Metzlaff (1990). Leaf tissue from node 3 or 4 (1.6 g) was ground to a fine powder in liquid nitrogen and added to 10 mL of lysis buffer (50 mM Tris-HCl, pH 7.6, containing 100 mM NaCl, 50 mM EDTA, and 0.5% SDS) and 10 mL
of 100 mM Tris-buffered phenol, pH 8.0. After the sample
was shaken vigorously, 5 mL of chloroform:isoamyl alcohol (24:1) was
added, the mixture was shaken further, the cell debris was pelleted by
centrifugation at 10,000g for 10 min at 4°C, and the
supernatant was extracted again with phenol and chloroform:isoamyl
alcohol. The aqueous phase was obtained by centrifugation at
10,000g for 10 min at 4°C; 0.67 volume of isopropanol was
added to precipitate the nucleic acids; the precipitate was collected
by centrifugation at 10,000g for 10 min at 4°C; and the
pellet was washed with 80% (v/v) ethanol and then dried at 40°C for
5 min. The pellet was resuspended in 4 mL of 10 mg mL 1 RNase in 10 mM
Tris-HCl, pH 8.0, containing 100 mM NaCl and 1 mM EDTA, and incubated at 37°C for 25 min
before extraction with 2 mL of phenol and 2 mL of chloroform:isoamyl
alcohol. The aqueous fraction was obtained by centrifugation at
10,000g for 10 min at 4°C; the nucleic acids were
precipitated with 2.5 volumes of 99.7% (v/v) ethanol; the precipitate
was collected by centrifugation at 10,000g for 10 min at
4°C; and the pellet was washed with 80% (v/v) ethanol, dried, and
then resuspended in 500 µL of sterile ultrapure water (Milli-Q,
Millipore). A260 was used to quantify DNA.
RNA was extracted essentially as described by Van Slogteren et al.
(1983). Frozen tissue was powdered under liquid nitrogen and added to 5 volumes of 100 mM Tris-HCl, pH 8.0, containing 100 mM LiCl, 10 mM EDTA, and 1% (w/v) SDS made up
as a 1:1 ratio with 100 mM Tris-buffered phenol, pH 8.0. After vortexing and the addition of 2.5 volumes of chloroform/isoamyl
alcohol (24:1), the extract was incubated at 50°C for 15 min. After
centrifugation at 5000g for 30 min at 4°C, RNA was
precipitated from the supernatant by overnight incubation in a final
concentration of 2 M LiCl at 4°C. The RNA was
collected by centrifugation as described before, extracted once more
with chloroform:isoamyl alcohol, and ethanol precipitated, and the
pellet was dried and then resuspended in water. To isolate
poly(A+) mRNA, the PolyATract system (Promega)
was used according to the manufacturer's instructions.
A260 was used to quantify
poly(A+) mRNA.
RT-PCR Amplification and 3 RACE
To amplify putative ACC oxidase-coding sequences, first-strand
cDNA synthesis using total RNA isolated from apical and leaf tissues
was performed with RT (Superscript RNase-H, Boehringer Mannheim) and a
17-mer oligo(dT)-primer, according to the instructions supplied with
the product. PCR amplification was achieved using a high-fidelity
system (Expand, Boehringer Mannheim) with one round of 30 cycles (1 min
at 92°C, 1 min at 42°C, 1 min at 72°C, with a final extension for
10 min at 72°C) using a thermal cycler (PTC-200, MJ Research,
Watertown, MA) and ACOF1 and ACOR1 as primers (Table
I). One-hundred microliters was used as
the reaction volume and, at the conclusion of the first round, 1.0 µL
was removed and used as template in a second PCR round with identical
amplification conditions but using ACOF2 and ACOR2 as primers (Table
I). PCR-generated sequences were T/A cloned into pCRII vectors
(Invitrogen, San Diego, CA) and sequenced using an automated DNA
sequencer (model 377, PRISM, Applied Biosystems). The database was
searched using Blast-N (Altschul et al., 1990). Sequence alignments
(AlignPlus, Science & Educational Software, Durham, NC) compared
putative ACC oxidase sequences generated by RT-PCR.
To amplify the 3 UTRs from each distinct ACC oxidase gene identified
after RT-PCR of the coding region (described above), first-strand cDNA
synthesis was done as described previously but using an ADAP primer
(Table I) with a 20-mer oligo(dT)-extension. PCR amplification was
first used to clone the 3 UTR with a portion of the reading frame so
that the corresponding 3 UTR could be identified. PCR amplification
conditions were as described previously, except for an annealing
temperature of 55°C. One or two rounds of PCR amplification were
performed with primers specific for each ACC oxidase gene (Table I).
PCR-generated sequences were cloned and sequenced as described above.
To obtain 3-UTR-specific sequences, one round of PCR amplification was
performed (annealing at 55°C) using each reading frame 3-UTR
sequence cloned in pCRII as a template and using the primers specific
for each ACC oxidase gene listed in Table I.
Sequence Phylogeny
A majority-rule consensus tree
(neighbor-joining/observed-distances/100-bootstraps) was built using
the computer program PAUP (Phylogenetic Analysis Using Parsimony,
version 4.0, Sinaur Associates, Sunderland, MA). Sequences used to
construct the tree are identified by accession number in the legend to
Figure 6.
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| Figure 6.
Phylogenetic analysis of ACC oxidase amino acid
sequences from white clover and other ACC oxidases in the database. A
majority rule consensus tree
(neighbor-joining/observed-distances/100-bootstraps) was reconstructed
using the computer program PAUP. Bootstrap values are shown and the
accession number of each sequence is provided in the parentheses.  ,
Leaf senescence-associated sequence. AT: Arabidopsis
thaliana; CS1, 2, and 3: Cucumis sativus,
Cs-ACO1, 2, and 3; PV: Phaseolus vulgaris; PHO1, 2, and
3: Pelargonium hortorum, ACO1, 2 and 3; HA1, 2, and 3:
Helianthus annus, ACCO1, 2, and 3; NT: Nicotiana
tabacum; MD: Malus domestica; PS: Pisum
sativum; VR1 and 2: Vigna radiata, pVR-ACO1 and
2; AC: Actinidia chinensis; CM1, 2, and 3:
Cucumis melo, CM-ACO1, 2, and 3; BP: Betula
pendula; LE1, 2, and 3: Lycopersicon esculentum,
ACO1, 2, and 3; PHY1, 3, and 4: Petunia hybrida, ACO1,
3, and 4; OS: Oryza sativa.
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Southern and Northern Analyses
For genomic Southern analysis, DNA (20-µg aliquots) was first
digested with 100 units of HindIII, EcoRI, or
XbaI in a total volume of 200 µL for 16 h at 37°C,
at which time another 50 units was added and the digestion was
continued for another 4 h. The digested DNA was precipitated with
the addition of 30 µL of 8 M ammonium sulfate
and 1 mL of ethanol and incubated for 15 min at  80°C. The
precipitated DNA was collected by centrifugation at 13,000g
for 15 min at 4°C. The pellet was washed with 75% (v/v) ethanol,
dried, and resuspended in 20 µL of gel-loading buffer (0.2% [w/v]
SDS, 10% [v/v] glycerol, 0.01% [w/v] bromphenol blue, and 0.02 M EDTA, pH 8.0). The digested DNA was separated
through a 0.8% (w/v) agarose gel with TAE (40 mM
Tris-acetate and 2 mM EDTA, pH 8.0) as a running
buffer for 4 h at 100 V, stained in 1% (w/v) ethidium bromide for
20 min, depurinated in 0.25 M HCl for 30 min,
denatured in 0.4 M NaOH and 3 M NaCl for 1 h, and then washed in transfer
buffer (8 mM NaOH and 3 M
NaCl) for 15 min.
The DNA was transferred onto a nylon membrane (Hybond
N+, Amersham) for 16 h using the downward
alkaline-capillary method of Chomczynski (1992). After transfer, the
DNA was cross-linked onto the membrane with a UV cross-linker
(Stratalinker 2400, Stratagene), and the membrane was neutralized in 50 mM sodium phosphate buffer, pH 7.5. DNA probes (3-UTR
sequences) were produced by PCR amplification as described previously,
and each fragment was randomly labeled with
[ -32P] dATP using a DNA-labeling system
(Megaprime, Amersham) according to the manufacturer's instructions.
After purification through microcolumns (ProbeQuant G-50, Amersham),
the denatured labeled probes were added to membranes bathed in
hybridization solution (0.25 M sodium phosphate, pH 7.2, 7% [w/v] SDS, 1% [w/v] BSA, and 1 mM EDTA, pH 8.0;
Church and Gilbert, 1984) and hybridized for 16 h at 65°C.
Membranes were washed at 65°C for 20 min in 20 mM sodium
phosphate, pH 7.2, 0.5% (w/v) SDS, 0.5% (w/v) BSA, and 1 mM EDTA, pH 8.0, and then for 20 min in 2× SSPE (0.36 M NaCl, 20 mM sodium phosphate, and 2 mM EDTA) and 0.1% (w/v) SDS, then for 20 min in
0.2× SSPE and 0.1% (w/v) SDS, and finally for 30 to 60 min in 0.1×
SSPE and 0.1% (w/v) SDS. After washing, membranes were exposed to
Kodak XAR-5 film at 70°C.
Each 3 UTR was also subjected to Southern analysis. Fifty nanograms of
each sequence was separated for 90 min through a 1.2% (w/v) agarose
gel and then immediately transferred to a nylon membrane for 4 h,
and the membrane was probed and washed.
For northern analysis, 1.0 µg of poly(A+) mRNA
was denatured in 2.2 M formaldehyde by heating at 70°C
for 10 min and then cooling on ice; the denatured RNA was separated for
5 h at 80 V through a 1.0% (w/v) agarose gel, using 20 mM Mops, 50 mM sodium acetate, 10 mM EDTA, and 2.23 M formaldehyde as the running
buffer. At the conclusion of electrophoresis, the separated RNA was
transferred to a nylon membrane for 4 h, and the membrane was
again probed and washed.
Antibody Production and Western Analysis
The reading frame of the three TRACO sequences was obtained by PCR
amplification using two primers:
5-GGAATTCAAGCNTGYSANAAYTGGGGH-3 to provide a 5-EcoRI site
and 5-GGCAAGCTTYTCYTTNGCYTGRAAYTT-3 to provide a
3-HindIII site. The PCR-generated fragment was then directionally cloned into the EcoRI/HindIII
polylinker sites on the expression vector pPROEX-1 (Life Technologies),
and the construct was transformed into the
Escherichia coli strain TB-1 (Life Technologies). Induction
with isopropylthio- -galactoside and
purification of the recombinant protein using nickel-based affinity
chromatography was according to the protocol from the manufacturer
(Life Technologies). Amino acid sequencing of trypsin-generated
fragments, as described by Watson et al. (1998) confirmed that the
amino acid sequence of the recombinant protein matched the translated
sequence of each reading frame, and SDS-PAGE confirmed that the
recombinant proteins were the size calculated from the translated
sequence (data not shown).
To produce antibodies, 250 µg of the TRACO2 recombinant protein (for
rabbits) and 100 µg of the TRACO1 and TRACO3 recombinant proteins
(for rats) were dissolved in PBS, emulsified with an equal volume of
Freund's complete adjuvant, and immunized through several sites on
each animal's back. Booster injections with the protein emulsified in
Freund's incomplete adjuvant were administered at monthly intervals
over 3 months.
SDS-PAGE was carried out essentially as described by Laemmli (1970).
Western blotting and antibody detection were achieved using the method
of Towbin et al. (1979) as described by McManus et al. (1994). Whole
sera from both rat and rabbit were used at a dilution of 1:1000, with
alkaline phosphatase-linked secondary antibodies used for primary
antibody detection. The intensity of recognition was determined using
image analysis. To achieve this, the developed membranes were imaged
using a videocamera (model DXC-3000P, Sony, Tokyo), and the image was
captured on a personal computer using a color frame grabber
(Visionplus, Imaging Technology, Inc., Bedford, MA).
Two images were captured for each sample, one of the membrane and one
of the background baseboard. The background image was smoothed using a
25- × 25-pixel moving average, and for each band of product deposition
the maximum pixel and two minimum values were found. The minima mark
the front and back edges of the band and the pixel values between front
and back are summed to give the integrated density.
To determine the molecular mass of the immune-recognized proteins, 8%
to 15% or 10% to 20% polyacrylamide gradient gels were used with the
method outlined by Hames and Rickwood (1981).
ACC Oxidase Enzyme Assays
Leaf tissue, previously frozen in liquid nitrogen and powdered,
was extracted in 5 volumes of ice-cold 100 mM Tris-HCl, pH 7.5 containing 10% (v/v) glycerol, 2 mM DTT, and 30 mM ascorbate. The slurry was filtered through Miracloth
(Calbiochem) and centrifuged at 12,000g for 15 min at 4°C.
Solid ammonium sulfate was added to the supernatant to give a final
saturation of 30%, and the salt was dissolved while keeping the
mixture ice-cold (approximately 30 min). After the sample was
centrifuged at 12,000g for 10 min at 4°C, solid ammonium
sulfate was added to the supernatant to give a final saturation of
90%, and the salt was dissolved while keeping the mixture ice-cold
(approximately 60 min). The precipitated protein was then collected by
centrifugation at 12,000g for 15 min at 4°C. The pellet
was dissolved in 50 mM Tris-HCl, pH 7.5, containing 2 mM DTT, 30 mM
ascorbate, and 10% (v/v) glycerol, and the solution was desalted using
Sephadex G-25 (Amersham) equilibrated in the same buffer.
The protein concentration in these extracts was measured using the
method of Bradford (1976). To measure ACC oxidase activity, 0.2-mL
aliquots of the enzyme preparation were warmed to 30°C, mixed with
0.8 mL of preequilibrated (30°C) reaction buffer in 4.5-mL-capacity
vacuum tubes (Vacutainer, Becton-Dickinson) to give a final
concentration of 50 mM Tris-HCl, pH 7.5, containing 1 mM ACC, 10% (v/v) glycerol, 2 mM DTT, 30 mM ascorbate, 50 µM FeSO4, and 30 mM
NaHCO3. The tubes were sealed and shaken at
30°C for (usually) 20 min, and then 1.0 mL of the gas phase was
removed and the ethylene content was determined with a gas
chromatograph (model GC-8A, Shimadzu, Kyoto, Japan).
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RESULTS |
Changes in Chlorophyll Content, Ethylene Evolution, and ACC Content
during Leaf Development
Rooting at a single node and the subsequent outgrowth of the
stolon over a dry matrix produces the full program of leaf development along the stolon from initiation at the apex through maturation, senescence, and necrosis (Fig. 1A). It is
known that, if the root primordium at a node is in contact with a moist
substratum, it will grow out as a nodal root (Thomas, 1987). In the
field, this occurrence has a large impact on development in the white
clover plant, with the natural senescence of old sections of the stolon proceeding in waves that are interrupted by the presence of strongly rooted nodes (Sackville-Hamilton and Harper, 1989). The use of this
growth system, in which root initiation is inhibited, produces plants
in which the number of leaves attached to the stolon reaches a constant
number as the production rate is balanced by the senescence rate.
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| Figure 1.
A, Stages of leaf development along a single
stolon of white clover. B, Total chlorophyll ( ), ethylene evolution
(), and ACC content ( ) determined from leaves excised from single
stolons identical to that shown in A. Results are mean values ± SE; n = 5. FW, Fresh weight.
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As an indicator of leaf maturity, total chlorophyll (chlorophyll
a and b) content was determined for each leaf
(Fig. 1B). In the example shown, an increase in chlorophyll content was
observed in leaves excised from the apex to the node to leaf 3 (the
developing leaves), and then a constant (maximum) content was
observed in leaves excised from nodes 4 to 9 (leaves 4-9, mature green
leaf tissue). In leaves excised from nodes 10 to 16 (leaves 10-16), chlorophyll content decreased, causing green/yellow and then yellow leaf tissue. No measurements were made from desiccated yellow or
clearly necrotic tissues.
In vivo, two stages of significant ethylene production were observed
(Fig. 1B). The first was observed at the apex, which failed to reach a
minimum value by leaf 3. Minimum evolution of ethylene was observed
from leaves 4 to 10, broadly coinciding with the mature-green leaf
stage, after which a second stage of significant ethylene evolution was
observed. Here the rate of ethylene production gradually increased to
leaf 16 (Fig. 1B) and decreased again only in necrotic tissue (data not
shown). ACC content remained constant in developing and mature-green
tissues and then increased in senescent tissue (leaves 11-16, Fig.
1B), in concert with significant senescent-associated ethylene
production.
Isolation of TRACO1, TRACO2, and TRACO3 Gene Sequences
RT-PCR was used to amplify approximately 800-bp cDNA sequences
from the apex, leaf 4 (mature green), leaf 9 (onset of senescence), and
leaf 14 (senescent) using primers designed to conserve domains within
ACC oxidase genes sequenced from several other plant species. Sequencing of clones from each tissue revealed three distinct sequences
with homology to other ACC oxidase genes in the database, and these
were designated TRACO1, TRACO2, and TRACO3. To produce gene-specific
probes, 3 RACE was used to amplify the 3-UTR sequences, since
significant sequence diversity in ACC oxidase genes occurs within this
UTR (Fluhr and Mattoo, 1996).
The deduced amino acid sequences from the three ACC oxidase genes
amplified from white clover are shown as a comparison with a consensus
sequence devised from 23 other ACC oxidases (Kadyrzhanova et al., 1997)
(Fig. 2). Comparison of the
RT-PCR-generated sequences with the consensus revealed that the
translated white clover sequence makes up the majority of the reading
frame. The amino acid sequence is complete to the carboxy terminus but
begins at the conserved Ala, 27 amino acids downstream of the initial
Met.
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| Figure 2.
Alignment of the deduced ACC oxidase amino acid
sequences from white clover with a consensus (CONS.) ACC oxidase
sequence compiled by Kadyrzhanova et al. (1997). Uppercase letters in
the consensus sequence indicate complete agreement within the 23 ACC
oxidases compared, and lowercase letters represent the most frequently
occurring residue. Regions of complete alignment of the white clover
sequences with the consensus sequence are boxed.
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The homology comparison at the nucleotide level of the three ACC
oxidase DNA sequences in white clover leaf tissue is shown in Table
II, with homology values ranging from
75% to 84% for the regions of the coding frame amplified by RT-PCR.
Comparison of the 3 UTRs revealed homology values between 55% and
61%, which confirms the greater sequence divergence within the 3 UTR.
The nucleotide sequences corresponding to each 3 UTR from the three ACC oxidase DNA sequences from white clover were also compared (Fig.
3). In TRACO1 and TRACO2, single
near-upstream elements and far-upstream elements could be identified,
whereas two elements of each are observed in TRACO3. Near-upstream
elements and far-upstream elements are proposed to direct processing at
the poly(A+) site situated near the near-upstream
element motif (Rothnie, 1996). The occurrence of more than one near-
and far-upstream element motif in the 3 UTR of TRACO3 suggests that
differential processing of TRACO3 mRNA may occur. Furthermore, during
the course of PCR of the 3 UTR regions of TRACO3, two additional
truncated sequences (indicated by arrows in Fig. 3), each with an
attendant poly(A+) tail, were amplified (data not
shown). These truncated forms were not observed during the PCR
amplification of the 3 UTR regions from TRACO1 and TRACO2.
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Table II.
Nucleotide homology values between reading frame
ACO sequences from white clover generated by RT-PCR and the
corresponding 3 UTRs generated by 3 RACE
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| Figure 3.
Alignment of the nucleotide sequences from the 3
UTRs of TRACO1, TRACO2, and TRACO3. The nucleic acid sequences of the
primers used for PCR amplification are underlined. The arrows in TRACO3
denote the sites of poly(A+) attachment identified in two
truncated 3-UTR sequences amplified by PCR. FUE, Far-upstream element;
NUE, near-upstream element.
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The specificity of each (full-length) 3-UTR sequence as a probe for
use in molecular analysis was confirmed by Southern analysis (Fig.
4). Each 3-UTR probe hybridized only to
its corresponding sequence under the hybridization and washing
conditions used.
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| Figure 4.
Southern analysis showing the specificity of
3-UTR sequences derived from white clover ACO genes. Fifty nanograms
of the 3 UTR from TRACO1, TRACO2, and TRACO3 was separated through a
1.2% (w/v) agarose gel and blotted onto nylon membranes. Each membrane
was probed with the 32P-labeled 3 UTR of TRACO1, TRACO2,
or TRACO3. Details of hybridization and washing conditions are in
``Materials and Methods''.
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Genomic Southern analysis was used to confirm that each 3-UTR sequence
was complementary to distinct genes (Fig.
5). Genomic DNA was digested with
EcoRI, HindIII, and XbaI, and the
hybridization pattern was compared using each 3 UTR as a probe.
Markedly different hybridization patterns were obtained for each probe,
confirming that each of the ACC oxidase DNA sequences generated
from white clover by RT-PCR and 3 RACE are complementary to
distinct genes. White clover is an allotetraploid, comprising two
distinct diploid genomes (Williams, 1987). Therefore, multiple banding
patterns can arise through polymorphisms at alleles on one or both
genomes.
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| Figure 5.
Genomic Southern analysis of ACO genes in white
clover. DNA (20 µg) was digested with EcoRI (E),
HindIII (H), and XbaI (X), separated
through a 0.8% (w/v) agarose gel, and blotted onto nylon membranes.
Each membrane was probed with the 32P-labeled 3 UTR of
TRACO1 (A), TRACO2 (B), or TRACO3 (C). Details of hybridization and
washing conditions are described in ``Materials and Methods''.
|
|
Phylogenetic Comparison of White Clover ACC Oxidase Sequences
A phylogenetic tree was reconstructed from an alignment of the
deduced amino acid sequence from the white clover ACC oxidases with
other ACC oxidases in the database (Fig.
6). TRACO2, ACO1 from bean (PV, accession
no. AF053354), and TRACO3 clustered together strongly, with the closest
relationship between TRACO3 and the bean ACO1. Other
leaf-senescence-associated ACC oxidases are indicated in Figure 6: LE1,
ACO1 from tomato (Barry et al., 1996), and CM1, CM-ACO1 from melon
(Lasserre et al., 1996). However, TRACO3 is more closely related to
TRACO2 than to these other leaf-senescence-associated sequences. TRACO1
is most closely related to pPE8 (PS, accession no. P31239) from pea
(Peck et al., 1993), and the nearest neighbor to this grouping is
pVR-ACO1 from mung bean (VR1, accession no. U06046; Kim and Yang,
1994).
Expression of TRACO1, TRACO2, and TRACO3 during Leaf Development
Gene-specific probes comprising the 3 UTRs were used in northern
analysis to determine the constitutive expression of each ACC oxidase
gene (Fig. 7). TRACO1 is expressed almost
exclusively in the apex, with a much lower intensity of hybridization
discernible in leaf 1 and no detectable hybridization (using this
method) in leaf 2 or any other leaf along the stolon. TRACO2 is
detectable in the apex, shows maximal expression in leaves 1 to 2, and
then gradually decreases in intensity such that no discernible
expression can be observed by leaf 11. The expression of TRACO3 is
clearly detectable first in leaf 8, reaching maximum expression in
leaves 13 to 16. The expression of TRACO1, TRACO2, and TRACO3 in leaves excised from nodes older than 16 was not undertaken.
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| Figure 7.
Northern analysis of ACO gene expression during
leaf ontogeny in white clover. One microgram of poly(A+)
mRNA, isolated from leaves of the apex and each node, was reduced in
2.2 M formaldehyde, separated thorough a 1% (w/v) agarose
gel in 2.2 M formaldehyde, and then transferred onto nylon
membranes. Each membrane was probed with the 32P-labeled 3
UTR of TRACO1, TRACO2, or TRACO3. Details of hybridization and washing
conditions are described in ``Materials and Methods''.
|
|
Changes in ACO Enzyme Activity and Protein Accumulation during Leaf
Development
In vitro, ACO enzyme activity was detected in the apex and
increased to reach a maximum in leaves 4 to 9, after which the activity
steadily declined (Fig. 8A). The pattern
of ACO activity observed contrasts with the trend of ethylene evolution
that is virtually undetectable in leaves 4 to 9 but then increases
again (Fig. 1B).
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| Figure 8.
A, ACO activity in vitro in extracts from the apex
and leaves excised from each node. Results are mean values ± SE; n = 5. prot., Protein. B, Western
analysis of ACO protein accumulation in extracts from the apex and
leaves excised from specific nodes detected using antibodies raised
against TRACO1 and TRACO2. Antibody recognition was detected using
alkaline-phosphatase-linked secondary antibodies.
|
|
Antibodies were produced to translated proteins from each ACO gene, and
western analysis was used to determine the accumulation of each protein
(Fig. 8B). Antibodies raised against TRACO1 recognized a protein of
approximately 205 kD (as determined by gradient SDS-PAGE), the
accumulation of which occurs predominantly in the apex, and then
declines until it is undetectable (using this method) after leaf 8. Antibodies raised against TRACO2 recognized a protein of 36.4 kD (as
determined by gradient SDS-PAGE), the accumulation of which broadly
matched the detectable ACC oxidase activity observed in Figure 8A. The
pattern of ACO accumulation detected by TRACO2 is seen most clearly
using image analysis to quantify signal intensity (Fig. 8C).
Antibodies raised against TRACO3 did not recognize any proteins in the
leaf extracts examined (data not shown). Weak recognition by TRACO3
antibodies (determined by a significantly longer development time in an
alkaline phosphatase substrate) was observed from a 36.4-kD protein
that had the identical accumulation pattern as that observed when using
antibodies raised against TRACO2 (data not shown). These observations
suggest that the antibodies raised against TRACO2 and TRACO3 recognize
the same protein, and each antibody recognizes the protein product of
the other three ACO gene products obtained using expression vectors in
E. coli.
| |
DISCUSSION |
In this study we have shown that three distinct ACC oxidase genes
are expressed differentially during leaf ontogeny in white clover. The
maximal expression of two of these genes coincides with the two peaks
of ethylene evolution observed. The expression of TRACO1 is
predominantly in the apex, whereas the expression of TRACO3 almost
precisely matches the increase in ethylene evolution during leaf
senescence.
Ethylene evolution from the apex has been reported in several plant
species (for review, see Osborne, 1991), with some consensus that in
dicotyledonous plants the role of the hormone is to limit cell
expansion in younger leaves (Osborne, 1991; Kieber et al., 1993; Lee
and Reid, 1996). However, to our knowledge, this is the first report of
apex-specific ACC oxidase gene expression. A scanning electron
microscope study of the apex of white clover revealed that it is a
complex tissue comprising the apical meristem, leaf primordia, and an
axillary bud at the third node (Thomas, 1987). As yet we cannot say
which of these tissues that comprise the apex specifically express
TRACO1, but tissue localization of expression using in situ
hybridization should provide significant clues regarding the role of
ethylene. The use of a phylogenetic tree has placed the apex-specific
sequence closest to pPE8, a cDNA clone isolated from pea seedling shoot
tissue (Peck et al., 1993). The next nearest neighbor was pVR-ACO1,
which is constitutively expressed in hypocotyl, leaf, and stem tissues
from mungbean seedlings (Kim and Yang, 1994). However, it was not
reported in either study whether these sequences were expressed
specifically in apical tissues.
Ethylene evolution from senescent leaves is now well documented, and
senescent leaves of many species can convert ACC to ethylene (Roberts
et al., 1985; Osborne, 1991). However, there are fewer reports of ACC
oxidase gene expression during leaf senescence. In tomato leaves
ethylene evolution increases as senescence proceeds, an increase that
coincides with an increase in transcript accumulation of the ACC
oxidase gene ACO1 (John et al., 1995).
More recently, a second ACO gene, ACO2, was shown to be
expressed at the onset of leaf senescence (Barry et al., 1996). A similar pattern in which two genes of ACC oxidase are differentially expressed in mature-green and senescent leaf tissue was also reported for melon (Lasserre et al., 1997). The differential pattern of gene
expression observed in white clover leaves is similar to these species.
However, the construction of a phylogenetic tree has revealed that the
white clover senescence-associated transcript (TRACO3) is more closely
related to TRACO2 than sequences expressed in senescent leaf tissue
from other species. TRACO3 is most closely related to PV-ACO1, a cDNA
cloned from bean seedling tissues whose expression is regulated by
light (Pidgeon et al., 1997); the authors did not report whether
this sequence is also expressed in senescent tissue.
The examination of ACO gene expression has been extended in white
clover with the measurement of corresponding ACC oxidase activity in
vitro during leaf ontogeny. Detectable ACC oxidase activity coincides
more closely with TRACO2 gene expression. Some ACO activity is observed
in the apex, but in leaves from nodes 13 to 16, where the expression of
TRACO3 is induced, there is no concomitant increase in detectable
enzyme activity in vitro. Leaf tissues from many species have been
shown to convert ACC to ethylene, evidence that an ACC-dependent
ethylene-forming system is functional in vivo (Osborne, 1991). However,
we are not aware of any studies in which ACO activity has been
demonstrated in vitro in senescent leaf extracts.
To determine the pattern of ACO protein accumulation (which may
be independent of detectable enzyme activity), antibodies were raised
to each gene product. In these experiments, the antibodies raised
against TRACO2 identified an ACC oxidase protein of 36.4 kD, which is
within the size range (35-41 kD) reported for ACC oxidase proteins
from other plant species (Dong et al., 1992; Dupille et al., 1993;
Pirrung et al., 1993; Rombaldi et al., 1994), and the relative
accumulation of the protein determined by western blotting matched the
measurable ACO activity in vitro. The TRACO1 antibody recognized a
protein of approximately 205 kD that was expressed predominantly in the
apex. Clearly, a protein of 205 kD cannot be transcribed from a 1.35-kb
mRNA transcript, although we cannot exclude the possibility that the
protein may be highly anomalous in terms of its migration using
SDS-PAGE. However, the 205-kD protein does warrant further
investigation because, although proteins of this size that exhibit ACC
oxidase activity have not been characterized previously, the
protein-accumulation pattern coincides with TRACO1 gene expression, and
neither the serum raised against TRACO2 nor the one raised against
TRACO3 recognized this protein. We are currently using immunoaffinity
approaches to purify a sufficient quantity of this protein for further
analysis.
In this study we were not able to demonstrate protein accumulation by
antibody staining in the senescent leaf tissue that coincides with
TRACO3 transcript accumulation. However, using hydrophobic and
ion-exchange column chromatography, preliminary purification of ACO
from senescent leaf tissue of white clover revealed a protein with ACO
activity that was distinct from an isoform purified from mature-green
leaf tissue (D. Gong and M.T. McManus, unpublished data). The apparent
unmasking of significant activity in senescence extracts after
hydrophobic column chromatography suggests that the activity of the
enzyme may be regulated quite differently in senescent tissue. Although
we have examined variables such as cofactor requirements and pH optima
in senescent extracts and found no significant difference compared with
mature-green extracts, such differential regulation of ACO activity has
been reported from experiments with corn and sunflower seedlings, in which the occurrence of organ-specific enzymes with different substrate
requirements has been demonstrated (Finlayson et al., 1997).
The results presented here add to the growing number of studies
demonstrating that the expression of the ACC oxidase gene family is
highly regulated during plant development. Furthermore, they show that
the two peaks of ethylene production during leaf ontogeny coincide with
the expression of distinct ACC oxidase genes. It has been shown that in
bean ethylene can induce the differentiation of its own target cell
class (McManus et al., 1998). Given that the ethylene produced at each
leaf developmental stage in white clover induces separate responses
(modulation of leaf growth in the apex and regulation of senescence in
mature tissues), it is interesting to speculate further that regulation of the biosynthesis of ethylene may be intimately linked to its competence to respond to it. A study of the molecular basis for the
control of transcription for each member, like the one done in melon
(Lasserre et al., 1997), will play an important part in establishing
such a link.
| |
FOOTNOTES |
1
This work was funded by the New Zealand
Foundation for Research, Science and Technology (contract no. C10 635),
by the New Zealand Agricultural and Pastoral Research Institute
(AgResearch) with provision of a Ph.D. study award to D.H., and by the
New Zealand Crop and Food Research Institute with provision of a Ph.D. study award to S.M.B.
2
These authors contributed equally to this work.
*
Corresponding author; e-mail m.t.mcmanus{at}massey.ac.nz; fax
64-6-350-5694.
Received August 24, 1998;
accepted December 23, 1998.
| |
ABBREVIATIONS |
Abbreviations:
RACE, rapid amplification of cDNA ends.
RT, reverse transcriptase.
UTR, untranslated region.
| |
ACKNOWLEDGMENTS |
We thank Professor S.F. Yang and Dr. Alan D. Campbell
(Department of Vegetable Crops, University of Davis, CA) for providing ACC oxidase primers and for help with the use of RT-PCR to generate the
reading frame sequences for TRACO2 and TRACO3; Lorraine Berry (Institute of Molecular Biosciences, Massey University, Palmerston North, New Zealand) and Catriona Knight (School of Biological Sciences,
Auckland University, New Zealand) for the amino acid sequencing; Dr.
Peter Lockhart (Institute of Molecular BioSciences, Massey University)
for help with reconstructing the phylogenetic tree; and Dr. David
Swofford (Smithsonian Institution, Washington, DC) for the prerelease
version of PAUP.
GenBank accession numbers for the sequences reported in this study are
AF115261 (TRACO1), AF115262 (TRACO2), and 115263 (TRACO3).
| |
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Top
Abstract
Introduction