ARTICLE IN PRESS

Journal of Plant Physiology 166 (2009) 333—343

www.elsevier.de/jplph

Fatty acid a-dioxygenase from Pisum sativum:

Temporal and spatial regulation during germination
and plant development
Anke K. Meisnera,1, Alexander Safferta,2, Peter Schreiera,, Astrid Schönb

a
Institute of Pharmacy and Food Chemistry, University of Würzburg, Am Hubland, 97074 Würzburg, Germany
b
University of Leipzig, Molecular Cell Therapy, Center of Biotechnology and Biomedicine, Deutscher Platz 5, 04103
Leipzig, Germany

Received 20 March 2008; received in revised form 29 May 2008; accepted 29 May 2008

KEYWORDS Summary
a-Oxidation;
a-Dioxygenases are expressed in plants in response to biotic and abiotic stress. They
Fatty acid
catalyze the enantioselective 2-hydroperoxidation of long-chain fatty acids, the
metabolism;
initial step of the a-oxidation pathway of fatty acids in plants. In this study, the
Germination;
complete cDNA of an a-dioxygenase from germinating pea seeds (Pisum sativum) is
Phyto-oxylipins;
presented. The deduced amino acid sequence establishes that the enzyme belongs
Plant defense
to the recently characterized family of a-dioxygenating enzymes in plants. We also
mechanism
present the first systematic study on the expression of a-dioxygenase in germinating
and developing pea plants. During germination, a-dioxygenase mRNA accumulates in
the cotyledons and the embryonic axis of pea seeds de novo. In developing pea
plants, the transcript is detected almost exclusively in roots. The accumulation of
a-dioxygenase protein parallels transcript accumulation in that it is abundant in
germinating as well as young plant tissue, and correlates with loss of mRNA during
plant maturation. a-Dioxygenase enzymatic activity in plant extracts is highest in
cotyledons during imbibition. In the embryonic axis and roots of developing plants
comparable activity levels are observed, whereas in shoots little a-oxidation activity
is detected. With this contribution, we present information on the temporal and
spatial expression of a-dioxygenase during plant germination and development,
supporting the hypothesis that the a-oxidation pathway of fatty acids plays a role
during plant developmental processes.
& 2008 Published by Elsevier GmbH.

Abbreviations: a-DOX, a-dioxygenase; LOX, lipoxygenase.

Corresponding author. Tel.: +49 931 8885480; fax: +49 931 8885484.
E-mail address: schreier@pzlc.uni-wuerzburg.de (P. Schreier).
1
Present address: Bundesamt für Verbraucherschutz und Lebensmittelsicherheit, Mauerstr. 39-42, 10117 Berlin, Germany.
2
Present address: Tetra Pak GmbH, International Packaging Research Laboratories, Untere Waldplätze 27, 70569 Stuttgart, Germany.

0176-1617/$ - see front matter & 2008 Published by Elsevier GmbH.

doi:10.1016/j.jplph.2008.05.009
 ARTICLE IN PRESS
334
Introduction
Phyto-oxylipins, a group of oxygenated fatty acid
derivatives, play a vital role in plant biology. They
are best known for their impact during plant
defense reactions; compounds generated by fatty
acid oxygenation possess signaling activity, e.g.
jasmonic acid and C-6 aldehydes, or antibacterial
and antifungal activity. Oxylipins are also known to
be involved in plant developmental processes.
(Blée, 2002; Wasternack, 2007).
The a-oxidation of fatty acids is the pathway by
which 2-hydroxy fatty acids, a group of oxylipins,
are synthesized. The a-oxidation of fatty acids was
first discovered in germinating peanut seeds (New-
comb and Stumpf, 1952) and has later been
observed in several other higher plants such as
pea leaves (Hitchcock and James, 1966), cucum-
bers (Galliard and Matthew, 1976) and potatoes
(Laties and Hoelle, 1967), but also in simple
organisms like the marine green algae Ulva pertusa
(Kajiwara et al., 1988). Several stable reaction
products of the a-oxidation pathway have been
detected, i.e. the respective 2-hydroxy fatty acid,
CO2 and a Cn1 aldehyde as well as the correspond-
ing chain-shortened fatty acid. Evidence for the
intermediary formation of an unstable 2-hydroper-
oxy acid has been provided by the detection of (R)-
2-hydroperoxypalmitic acid as the first reaction
product of the a-oxidation pathway (Akakabe
et al., 1999). The 2-hydroperoxy fatty acids were
shown to decarboxylate spontaneously, leading to
the formation of the chain-shortened Cn1 alde-
hyde (Hamberg et al., 1999).
The characterization of the family of enzymes
that catalyze the initial a-dioxygenation step of the
a-oxidation pathway, i.e. the enantioselective
introduction of O2 at the C-2 position of fatty acids
leading to the formation of unstable (R)-2-hydro-
peroxy fatty acids, was accomplished in 1998 by the
identification of a pathogen inducible oxygenase
(PIOX, later renamed a-DOX for a-dioxygenase)
from tobacco leaves and a homologous enzyme
from Arabidopsis thaliana, both of which are
capable of catalyzing the conversion of fatty acids
to their corresponding 2-hydroperoxides (Sanz
et al., 1998; Hamberg et al., 1999). In the mean-
time, the occurrence of fatty acid a-dioxygenating
enzymes has been shown for other higher plants,
e.g. Pisum sativum (Saffert et al., 2000) and Oryza
sativa (Koeduka et al., 2002). Expression of a-DOX
genes in plants has been shown to be induced in
response to abiotic and biotic stress, including
bacterial infection (Sanz et al., 1998; Kim et al.,
2002), cellular signals mediating cell death (Ponce
de León et al., 2002), herbivore attack (Hermsmeier
A.K. Meisner et al.
et al., 2001), high-salinity stress (Wei et al., 2000),
wounding (Tirajoh et al., 2005) and heavy metal
stress (Koeduka et al., 2005). Apart from the
expression of a-DOXs during the plant response to
stress, the a-oxidation of fatty acids is detected
during developmental processes, such as plant
germination. We reported previously that a-DOX
activity is markedly elevated in germinating pea
seeds compared to leaves of mature plants (Saffert
et al., 2000), suggesting that the a-oxidation
pathway plays a role during the plant germination
process.
In this contribution, we present the complete
cDNA sequence of a-DOX from germinating pea
seeds, as well as our results on a-DOX transcript and
protein accumulation, and the time-dependent
changes in fatty acid a-dioxygenating activity in
pea seedlings and young pea plants. Taking our
results into consideration, the role a-dioxygenation
might play in different tissues during plant germi-
nation is discussed.
Materials and methods
Plant material
All studies were performed with P. sativum L. cv.
‘‘Kleine Rheinländerin’’. Dry seeds were surface ster-
ilized in 4% sodium hypochlorite solution and grown
hydroponically in sterile expanded clay under a 14 h
light/10 h dark photoperiod at 22 1C in a growth chamber.
Plants were irrigated with a half-strength solution of
Hoagland’s no. 2 Basal Salt Mixture (Sigma). Plant age
was defined according to the time of growth after the
start of imbibition. At 6, 12 and 24 h of imbibition the
embryonic axis and the cotyledons were collected
separately. At 3, 6, 9, 12, 16, 20, 24 and 28 d after the
start of hydration the growth axis was separated into
roots and shoots. All samples were shock frozen in liquid
nitrogen immediately after collection and stored at
80 1C.
Amino acid sequencing
Amino acid sequencing of internal fragments of
purified a-DOX from germinating pea seeds was per-
formed as described previously (Saffert et al., 2000).
cDNA synthesis
Total RNA was isolated from pea seeds after 3 d
of germination according to the method of Logemann
et al. (1987). Poly(A)+ RNA was isolated with
oligo(dT) cellulose. Complementary DNA was synthesized
using a mix of anchored oligo(dT) primers (dT16A,
dT16G, dT16C). Two degenerate oligonucleotide primers
(DOX1A 50 -ACT-AAG-CAG-GTT-GAR-CTT-AGY-GC-30 and
 ARTICLE IN PRESS
Expression of a-dioxygenase in pea plants
DOX4B 50 -CCA-CAI-GCT-TGG-TGA-CCC-ATT-GA-30 ) were
constructed on the basis of the partial amino acid
sequences of the 70 kDa subunit of purified pea a-DOX,
taking into account highly conserved regions of plant a-
DOX sequences, and used for PCR amplification. The
resulting PCR product with a length of 830 bp (DOX1A/4B)
was cloned into the Sma I restriction site of pUC19 to give
plasmid pDOX1A/4B and sequenced. The fragment was
recovered and labeled radioactively for use as a probe for
cDNA library screening and RNA gel blot hybridization.
cDNA library construction and screening
Poly(A)+ RNA was prepared from total RNA isolated
from germinating pea seedlings as described above.
Complementary DNA was synthesized and cloned into
the EcoR I-Xho I sites of the Uni-ZAPs XR vector
(Stratagene), following the manufacturer’s instructions.
The ligation was packaged using a ZAP-cDNAs Gigapacks
III Gold Cloning kit (Stratagene). Approximately 109
clones were screened with the 32P-labelled DNA probe
of pea a-DOX generated from pDOX1A/4B. Transfer of
bacteriophage l plaques to nitrocellulose filters and
subsequent immobilization was performed according to
Sambrook et al. (1989). Nitrocellulose filters were
hybridized overnight at 50 1C in 50% (v/v) formamide,
5  SSC, 20 mM sodium pyrophosphate, 0.1% (w/v) SDS,
1  Denhardt’s solution (0.02% (w/v) Ficoll, 0.02% (w/v)
polyvinylpyrrolidone, 0.02% (w/v) bovine serum albu-
mine, 100 mg mL1 denatured salmon DNA). After hybri-
dization, blots were washed twice with 5  SSC, 0.1%
(w/v) SDS for 10 min at room temperature, once with
1  SSC, 0.1% (w/v) SDS for 10 min at room temperature,
once with 0.1  SSC, 0.1% (w/v) SDS at room tempera-
ture; the washing temperature subsequently was raised
step-wise up to 50 1C. Filters were exposed to sensitized
X-ray Retina XBD with an intensifying screen at 60 1C.
Positive clones were picked and the pBluescript SK(+/)
phagemid excised with the helper phage and recircular-
ized. The clone with the longest cDNA insertion covering
the full length open reading frame was sequenced
completely (clone DOX8-7/A).
Analysis of gene expression
Total RNA was isolated from plant samples with the
RNeasy Plant Mini Kit (Qiagen) according to the manu-
facturer’s instructions. For the isolation of total RNA from
dry seeds and cotyledons the buffer RFC was used, and
for all other applications the buffer RPE was used. On-
column digestion of DNA was performed with RNase-free
DNase I. Total RNA (10 mg) was separated electrophor-
etically on 1% (w/v) agarose-formaldehyde gels and
transferred to a positively charged nylon membrane by
upward capillary transfer with 10  SSC as transfer
buffer. The amount of loaded RNA was documented by
ethidium bromide staining. DNA probes were generated
as described above. Blots were hybridized at 65 1C in
Rapid-hyb Buffer (Amersham Biosciences). After hybridi-
zation, membranes were washed twice with 2  SSC, 1%
335
(w/v) SDS for 15 min at room temperature and consecu-
tively at 40, 50 and 60 1C, and finally once with 1  SSC,
0.5% (w/v) SDS for 15 min at 60 1C. Membranes were
exposed to X-ray Retina XBD with an intensifying screen
at 60 1C and subsequently to PhosphoImager plates
(Molecular Dynamics).
Antibody production and immunoblot analysis
Polyclonal antibodies against a-DOX from germinating
pea seeds were raised in rabbits by injecting the 70 kDa
subunit of the pea a-oxidation enzyme. Following
purification carried out as described previously (Saffert
et al., 2000), fractions from Mono-P HR5/20 were
separated by native polyacrylamide gel electrophoresis.
A single protein band of 230 kDa was detected by staining
with Coomassie Brilliant Blue. The protein band of
230 kDa was excised from the gel and the respective
protein was electroeluted from the gel matrix. The
eluted protein was subsequently separated by SDS-PAGE,
and the resulting 70 kDa subunit was used for antibody
production.
Rabbit antisera often contain immunoglobulins that
recognize bacterial antigens and thus may lead to
nonspecific background signals in immunoblots. To reduce
the amount of immunoglobulins recognizing bacterial
antigens, the antiserum was incubated with insoluble
material of inactivated Escherichia coli cells and used for
immunoblots after removal of E. coli material and bound
immunoglobulins by centrifugation as described by
Gruber and Zingales (1995).
Plant samples were collected at the indicated times of
growth and frozen in liquid nitrogen. Plant material was
ground to a fine powder in liquid nitrogen. Extraction
buffer (0.1 M Tris–HCl, pH 7.5) was added at a ratio of
2 mL g1 fresh weight and samples were homogenized.
The suspensions were clarified by centrifugation for
10 min at 3000g. A total of 50 mg of total protein were
separated on 10% (w/v) SDS-PAGE gels and transferred to
nitrocellulose membranes by electroblotting. After the
transfer, membranes were washed in TBS (pH 8.0) and
blocked overnight at 4 1C in a solution of 0.2% (w/v)
I-Block (Tropix) in TBS. Subsequently blots were incu-
bated for 5 h at room temperature in blocking solution
with anti-dioxygenase antiserum (1:1000, purified against
E. coli antigens as described above). Thereafter, mem-
branes were washed twice (10 min at room temperature)
in blocking solution and incubated with a horseradish
peroxidase-conjugated secondary antibody (Sigma) di-
luted in blocking solution (1:5000) for 1 h at room
temperature. Antigen–antibody complexes were visua-
lized with Luminol as chemiluminescent substrate.
Enzyme assay
Plant samples were harvested at the times indicated
and frozen in liquid nitrogen. Protein extraction was
performed as described above. After centrifugation
protein extracts were used for the determination of
a-DOX activity. Aliquots were incubated with 1 mM
 ARTICLE IN PRESS
336 A.K. Meisner et al.

palmitic acid for 30 min at 30 1C. After addition of a
saturated acidic solution of 2,4-dinitrophenylhydrazine in
ethanol and subsequent extraction with hexane, penta-
decanal formed during the incubation was analyzed as its
2,4-dinitrophenylhydrazone by reversed.
Phase HPLC analysis and UV detection at 350 nm. 2,4-
Dinitrophenylhydrazones were eluted using a binary
gradient system with a flow rate of 1 mL min1. The
gradient proceeded from 95% (v/v) water acidified with
0.05% (v/v) formic acid and 5% (v/v) acetonitrile to 100%
acetonitrile in 20 min and from 100% acetonitrile to 30%
(v/v) acetonitrile and 70% (v/v) 2-propanol in 10 min. For
the quantification of pentadecanal, calibration curves
were generated with the authentic reference synthesized
as described earlier (Saffert et al., 2000).
Results
Amino acid sequencing of a-DOX purified
from pea and cDNA isolation
Sequences corresponding to the remaining clones
were shorter, but identical in regions present in all
clones. The complete cDNA sequence (sequence
data have been deposited at GenBank under
accession number AJ784963) comprises an open
reading frame of 1929 bp in length. The deduced
polypeptide of 643 amino acids has a calculated
molecular mass of 73.3 kDa, with its sequence being
in agreement with the partial amino acid sequences
of the 70 kDa subunit of the a-DOX purified from
pea (Figure 1). Sequence alignments with known
plant fatty acid a-DOXs revealed high similarity on
the amino acid level. The highest sequence identity
(75% of identical amino acids) was found with the
a-DOX1 from N. tabacum (GenBank accession
number AJ007630), and sequence alignments with
the A. thaliana a-DOX1 (accession number
AF334402) revealed 72% identity at the amino acid

As described earlier, the a-oxidation enzyme

purified from germinating pea seeds has a mole-
cular mass of about 230 kDa with subunits of 70 and
50 kDa, a-DOX activity being associated with the
70 kDa subunit. The amino acid sequences of two
internal fragments of the 70 kDa subunit, generated
by cyanogen bromide cleavage, showed highly
significant similarity to the amino acid sequence
of the a-DOX from Nicotiana tabacum (Saffert
et al., 2000). Sequencing of further internal
fragments of the purified enzyme from pea sup-
ported the finding that it is a member of the family
of a-DOX enzymes in plants. For the isolation of the
a-DOX cDNA from pea, poly(A)+ RNA was isolated
from germinating seeds after 3 d of growth, at a
time when a-oxidation activity has been shown to
be high (Saffert et al., 2000) and, in consequence,
the amount of a-DOX transcript could be expected
to reach high levels. After reverse transcription of
poly(A)+ RNA with oligo(dT) primers, a part of the
sequence encoding the pea a-DOX was isolated by
PCR. Degenerate oligonucleotide primers for the
amplification were constructed on the basis of the
amino acid sequences of internal fragments of
the purified pea a-DOX, taking into account highly Figure 1. Sequence alignment of the amino acid
conserved regions in plant a-DOX sequences. A PCR sequences of internal fragments of the 70 kDa subunit
product with a length of 830 bp (DOX1A/4B) was of purified a-dioxygenase from germinating pea seeds (Ps
obtained. This fragment of the pea a-DOX cDNA pep; X indicating amino acids that could not be identified
due to technical problems) and the translated sequence
served as a probe to screen approximately 109
of the complete cDNA of P. sativum a-dioxygenase (Ps
clones of a cDNA library prepared with poly(A)+ RNA DOX). Identical amino acids are shaded in black. Black
isolated from germinating pea seeds. Eventually, 11 bars 1A and 4B indicate the positions of the PCR
positive clones were isolated and analyzed by amplification primers DOX1A and DOX4B. Amino acid
sequencing. The clone with the longest cDNA residues that have been shown to be catalytically
insertion, providing the full length open reading relevant in other plant a-dioxygenases are marked by
frame, was sequenced completely (clone DOX8-7/A). an asterisk (His-168, Tyr-390 and His-393).
 ARTICLE IN PRESS
Expression of a-dioxygenase in pea plants 337

level. Comparison of the pea a-DOX to the feebly--

like protein of A. thaliana (accession number
AY128743), designated as a-DOX2 (Hamberg et al.,
2002), revealed 62% identical amino acid residues.

Analysis of gene expression during

germination and development

To gain information on the role fatty acid a-

dioxygenation might play during plant germination,
we examined the expression of P. sativum a-DOX
during the germination process. For this purpose,
the amount of a-DOX mRNA in different tissues of
pea plants was determined at various time points
during the germination of pea seeds and during the
growth of young pea plants. To demonstrate
differences in temporal and spatial distribution,
the expression was analyzed differentially in dry
seeds, the embryonic axis and the cotyledons
during the first day after the start of imbibition as
well as in root and shoot tissue of healthy plants on
the third and following days of growth. RNA gel blot
analyses were performed with equal amounts of
total RNA isolated from plant samples collected at
defined time points. Probing with the P. sativum a- Figure 3. RNA gel blot analysis of a-dioxygenase mRNA
DOX cDNA revealed that a-DOX transcript was expression in developing pea plants. Samples obtained
abundant during germination. However, the pat- from roots and shoots were analyzed. Analysis of P.
tern of mRNA accumulation differed considerably in sativum total RNA was performed as in Figure 2.
the analyzed tissues. As can be seen in Figure 2,
compared to dry seeds, a-DOX mRNA steadily of a-DOX mRNA was found at 12 and 24 h after the
accumulated in the embryonic axis as well as in start of hydration in the embryonic axis. After
the cotyledons on the first day after the start of the first day of growth, we exclusively examined the
imbibition. During the first day, the maximum level growth axis and analyzed roots and shoots sepa-
rately. The differentiation of the growth axis into
roots and shoots revealed that a-DOX transcript was
present mainly in roots and to a considerably lower
extent in shoots (Figure 3). The maximum level of
pea a-DOX mRNA in root material was found on the
third day of growth, and the transcript level
decreased continuously afterwards.

Immunoblot analysis of protein accumulation

during germination and development

To determine whether the pattern of transcript

Figure 2. RNA gel blot analysis of a-dioxygenase mRNA
expression in tissues of germinating pea seeds during the
first day of germination. Samples from dry seeds,
embryonic axes and cotyledons were analyzed. Total
RNA (10 mg) was hybridized with a 32P-labelled probe
derived from the pea a-dioxygenase cDNA. Ethidium
bromide stained gel before blotting is shown (bottom
panel).
accumulation of P. sativum a-DOX during seed
germination correlates with the level of a-DOX
protein in the respective plant tissues, we exam-
ined the abundance of the protein responsive to the
antiserum raised against the 70 kDa subunit of
purified a-DOX from germinating pea seeds. Protein
gel blot analyses were performed with equal
amounts of protein (50 mg of total protein) isolated
from plant samples harvested at defined time
points. a-DOX protein was detected in all analyzed
 ARTICLE IN PRESS
338
plant tissues. Similar to the pattern of mRNA
accumulation, a-DOX protein was abundant in
tissues of young plants, with decreasing amounts
in samples derived from developing plants. The
highest amounts of a-DOX protein were detected in
dry seeds as well as in cotyledons during the first
24 h of imbibition; a weaker signal was detected in
protein samples from the embryonic axis on the
first day of germination (Figure 4A). After the first
day of germination, we examined the growth axis,
which was separated into root and shoot. The
maximum amount of a-DOX protein was detected
on the third day of growth. But in contrast to the
Figure 4. Immunoblot analysis of a-dioxygenase protein
accumulation in different plant tissues. P. sativum
protein extracts (50 mg of total protein, separated by
SDS-PAGE) were probed with the antiserum raised against
the 70 kDa subunit of a-dioxygenase purified from pea.
The position of molecular mass markers is indicated on
the left. (A) Samples obtained from dry seeds, embryonic
axes and cotyledons during the first day of germination
were analyzed. (B) Analysis of samples obtained from
roots and shoots of pea plants.
A.K. Meisner et al.
findings from the RNA gel blot analyses, where the
time course of transcript accumulation showed a
pronounced distinction between the transcript
levels in root and in shoot tissue, a similar time
course was observed for root and shoot tissue with
regard to protein accumulation (Figure 4B). After
the third day of growth, the amount of detected
a-DOX protein in shoots and roots decreased to
reach a consistently low level around day 12 after
the start of imbibition.
Time-dependence of a-DOX activity in
germinating and developing pea plants
To evaluate the temporal development of the
a-DOX activity and its spatial distribution during the
germination of pea seeds, we analyzed a-DOX
activity by determining the conversion of palmitic
acid to pentadecanal by protein extracts from
different tissues of germinating and young pea
plants.
The pH-optimum of pea a-DOX lies between pH
6.8 and 8, peaking at pH 7.4 (Saffert et al., 2000).
Therefore, a pH of 7.5 was chosen to perform
activity assays. Palmitic acid has been shown to be
a suitable substrate for a-DOX from pea (Saffert,
2001). Taking into account the accessibility of
pentadecanal as reference substance, palmitic acid
was chosen as substrate for standard activity
assays. The incubation of crude homogenates of
young pea leaves and crude extracts of germinating
peas with fatty acids of different chain lengths
leads predominantly to the formation of the
respective chain-shortened aldehydes, the corre-
sponding 2-hydroxy fatty acids being formed to a
lesser extent (Adam et al., 1998). The initial
products of the a-oxidation pathway – the 2-
hydroperoxy fatty acids – are instable compounds.
It is therefore not feasible to quantify them in
order to determine a-DOX activity in standard
activity assays. The chain-shortened aldehyde,
the main product of the reaction, was quantified
to measure a-DOX activity. Pentadecanal formed
during the enzymatic reaction was analyzed by
reversed.
Phase HPLC as its 2,4-dinitrophenylhydrazine
derivative. a-DOX activity is expressed as nmol of
pentadecanal formed from palmitic acid per min
either per mg of total protein or per g of sample
fresh weight.
Again, we considered the dry seeds, the embryo-
nic axis and cotyledons on the first day after the
start of imbibition as well as the growth axis,
divided into roots and shoots, from day 3 to 28 of
growth (Figure 5). Of all analyzed plant samples,
 ARTICLE IN PRESS
Expression of a-dioxygenase in pea plants 339

Figure 5. Analysis of a-dioxygenase activity in different tissues of germinating pea seeds and developing pea plants. (A)
a-Dioxygenase activity, analyzed in dry seeds (black bars), the embryonic axis (light gray bars) and in cotyledons (dark
gray bars) of pea seeds on the first day of germination. Activity is expressed as nmol of pentadecanal formed min1 g1
of fresh weight. (B) a-Dioxygenase activity as in A but expressed as nmol of pentadecanal formed min1 mg1 of total
protein. (C) a-Dioxygenase activity, analyzed in roots (black bars) and shoots (light gray bars) of pea plants. Activity is
expressed as nmol of pentadecanal formed min1 g1 of fresh weight. (D) a-Dioxygenase activity as in C but expressed
as nmol of pentadecanal formed min1 mg1 of total protein. Each data point represents the values obtained in four
independent experiments. The mean and standard deviation are given.
 ARTICLE IN PRESS
340
the highest a-DOX activity per g of plant fresh
weight was found in the cotyledons at 24 h after
the start of imbibition. During the first day of
imbibition the a-DOX activity increased steadily
to the maximum at 24 h (95 nmol of pentadeca-
nal min1 g1 fresh weight). The activity in the
embryonic axis was less than half of the activity
in cotyledons and remained constant (37 nmol
of pentadecanal min1 g1 fresh weight). In the
embryonic axis as well as in the cotyledons, the
activity was higher than in dry seeds (Figure 5A).
The specific activity on the first day of germina-
tion ranged from 0.5 nmol of pentadeca-
nal min1 mg1 protein in dry seeds to 4 nmol of
pentadecanal min1 mg1 protein in cotyledons
(Figure 5B). These findings are in accordance with
the results of RNA and protein gel blot analyses.
When the growth axis was exclusively considered
after the first day of germination, the a-DOX
activity with reference to the fresh weight of
plant material was found to be generally higher in
roots than in shoot tissue. Activity levels in roots
with reference to the fresh weight remained
relatively constant over the observed time course
(peaking at days 12 and 16). In shoot tissue, the
activity was slightly increased on the first days
of growth and decreased subsequently to the
low level typical for healthy mature plants
(Figure 5C). It is noteworthy that, in shoots, no
significant variation of the a-DOX specific activity
(0.4–1.3 nmol of pentadecanal min1 mg1 pro-
tein) was observed during the time course. In roots,
however, an increase in the specific activity was
visible with a maximum specific activity of
18 nmol of pentadecanal min1 mg1 protein on
day 16 (Figure 5D). This increase in the specific a-
DOX activity in root extracts is not reflected in the
results on a-DOX mRNA and protein accumulation.
Discussion
An enzyme capable of a-dioxygenating fatty acid
substrates in the C-2 position has previously been
isolated from germinating pea seeds (Saffert et al.,
2000). We isolated the cDNA encoding the P.
sativum a-DOX, the deduced amino acid sequence
homologous to known plant a-DOXs. The P. sativum
a-DOX shares higher amino acid identity with the
enzyme a-DOX1 from A. thaliana than with the
putative a-DOX2 from A. thaliana (72% versus 62%),
indicating that the a-DOX from P. sativum belongs
to the a-DOX1 branch of the family of fatty acid
a-DOXs in plants. The amino acid residues sug-
gested to be catalytically relevant in a-DOX1 from
N. tabacum (His-167, Tyr-389 and His-392) by Sanz
A.K. Meisner et al.
et al. (1998) are conserved in the a-DOX from pea
(His-168, Tyr-390 and His-393) (Figure 1). The
corresponding residues have been shown to be
essential for the activity of a-DOX from rice
(Koeduka et al., 2002) and A. thaliana (Liu et al.,
2004).
In our study on a-DOX expression in germinating
and young pea plants, we show that germination
leads to a pronounced accumulation of a-DOX mRNA
in pea seeds and that a-DOX protein is abundant in
germinating and young pea plants. a-DOX mRNA
accumulated during imbibition in the embryonic
axis and cotyledons, indicating de novo transcrip-
tion. In the growth axis, a-DOX transcript was
detected almost exclusively in roots and to a
considerably lower extent in shoots. These results
are in accordance with the findings in mature
healthy A. thaliana plants, where a-DOX1 transcript
has been detected in the roots, but no transcript
has been found in leaves and stems (Ponce de León
et al., 2002).
Similar to transcript accumulation, a protein
immunoresponsive to antiserum raised against the
70 kDa subunit of the purified a-DOX from pea was
abundant in dry seeds and in imbibed cotyledons as
well as the embryonic axis. Unlike the time course
of transcript accumulation, that of protein accu-
mulation was comparable in roots and in shoots. It
is not known whether this is due to the existence of
different enzyme isoforms, which could possibly
have been detected by the antiserum and not the
DNA probe, or to a different regulation of a-DOX
expression in roots and shoots. It has also been
reported that, for some genes, mRNA levels do not
correlate well with protein levels, making it
difficult to predict protein expression from mRNA
levels (Gygi et al., 1999). Although it might be
conceivable that other pea proteins are recognized
nonspecifically by the antiserum used for the
immunoblots, taking into account the protein
purification as procedure, it must be considered
highly improbable that the antiserum is not specific
for a-DOX. To provide additional evidence, the
antiserum has been shown to recognize a-DOX from
pea expressed in E. coli and purified (data not
shown).
With respect to the temporal and spatial dis-
tribution of fatty acid a-dioxygenating activity
during the germination process and the growth of
young pea plants, a-dioxygenation of palmitic acid
increased markedly during the imbibition process.
When comparing a-DOX activity in roots and in
shoots normalized to the samples’ fresh weight, it
is noteworthy that activity levels in roots exceeded
those in shoots by 2–8 times. The low activity levels
in shoots are characteristic for healthy mature
 ARTICLE IN PRESS
Expression of a-dioxygenase in pea plants
plants and are in agreement with our previous
finding, that the rate of a-oxidation in pea leaves is
distinctly lower than in germinating seeds (Saffert
et al., 2000). The findings for a-dioxygenating
activity in germinating pea plants differ from the
results for a-DOX activity during the germination of
rice seedlings reported by Koeduka et al. (2002). In
rice seedlings, a-dioxygenation of fatty acids has
not been observed until a week after sowing, and
little activity has thereafter been detected in root
extracts, whereas in shoot extracts, the activity
levels have been found to rise steadily up to a 10-
fold increase in comparison to roots. These diver-
ging results may be attributable to differences in
the biology of monocotyledonous and dicotyledo-
nous plants, as differences in the regulation of
a-dioxygenating enzymes in rice, tobacco and
Arabidopsis have been reported as well (Koeduka
et al., 2005). When considering our results on
a-dioxygenating activity with respect to the
amount of total protein, a pronounced increase in
the specific activity was observed in roots during
maturation of the plants. This increase was not
reflected in the time course of transcript accumu-
lation or in the accumulation of immunoresponsive
protein. In Arabidopsis and tomato, the existence
of additional a-DOX isoforms has been postulated
(Hamberg et al., 2002; Tirajoh et al., 2005). Our
results suggest that in P. sativum different isoforms
of a-dioxygenating enzymes coexist as well. Possi-
bly, after the first days of development a shift from
the expression of pea a-DOX1 to other isoform(s)
takes place. It is also conceivable that the enzyme
activity is modulated by glycosylation or phosphor-
ylation of the protein. We have found that the
pea enzyme is glycosylated in vivo (data not
shown), but nothing is known about other protein
modifications or their possible regulatory signifi-
cance in this case.
Our observation that a-dioxygenating expression
was detected predominantly in the roots and
cotyledons of pea plants during plant germination
and development is plausible when we consider
that expression of a-DOX has been reported to
occur in plants in response to incidences associated
with cellular damage such as herbivore attack,
pathogen infection or senescence (Sanz et al.,
1998; Hermsmeier et al., 2001; Obregón et al.,
2001). Disruption of the cellular structure and cell
death is frequently correlated with the generation
of an oxidative burst which is known to be part of a
general stress defense pathway up-regulated by
biotic and abiotic stresses as well as during
different plant developmental processes such as
senescence. Roots consist mainly of lignified cells
that need to undergo cell death during lignification.
341
It has been noted that analogies exist between
processes in the lignifying xylem of Zinnia elegans
and the oxidative burst observed during the
hypersensitive plant cell response (Barcelo, 1999).
In cotyledons, where a-DOX protein, activity and
mRNA are highly abundant, the disorganization of
cellular structures allows the mobilization of
nutrients to be transported to the growing parts
of the plant. For the hypogaeic P. sativum it is well
known that the cotyledons undergo senescence
during germination as soon as the storage material
has been mobilized.
The reaction products of the a-oxidation pathway
belong to the oxylipins, a family of compounds
whose members are known to possess antimicrobial
activity (Prost et al., 2005). In planta, 2-hydroxy
fatty acids of differing chain lengths have been
characterized as the main products of the fatty
acid a-oxidation pathway and have been shown to
act during plant–pathogen interaction (Hamberg
et al., 2003). Since roots are continuously exposed to
soil bacteria, and are therefore prone to infection,
the preferential occurrence of a-oxidation processes
in roots could indicate a role for a-oxidation as a
permanent system of protection against infection in
roots. It has been shown that healthy transgenic
plants expressing defense-related genes prior to
pathogen infection become more resistant to certain
pathogens (Cao et al., 1998). Expression of a-DOX
during germination could thus contribute to improv-
ing the defensive potential of the plant during early
growth processes.
With the characterization of a-DOXs, a further
class of enzymes in plants capable of dioxygenating
fatty acids has been described in addition to
lipoxygenases (LOXs). a-DOXs as well as LOXs not
only catalyze related reactions, but show also
similarities in their expression pattern. They share
parallels with regard to the expression during
plant–pathogen interaction, but also with regard
to the expression during developmental processes
as senescence and germination. As has been
postulated for the family of a-DOXs, different
isoforms of LOXs exist in most, if not all, plant
species studied thus far. Young and expanding
tissues usually contain high levels of LOX enzymes,
but increases in senescing tissue have been
reported as well. The actual function of LOXs in
the process of germination remains unknown,
although several hypotheses have been put for-
ward, including nutrient mobilization, membrane
degradation or defense against pathogens (reviewed
in Porta and Rocha-Sosa, 2002; Liavonchanka and
Feussner, 2006).
With the present systematic study we provide
evidence that a-DOX mRNA and protein accumulate
 ARTICLE IN PRESS
342
during the germination of pea seeds. We have
shown specifically that a-dioxygenating activity is
highly abundant in the cotyledons of germinating
seeds. In healthy developing plants, a-DOX expres-
sion is detected preferentially in roots, with the
time course of a-dioxygenating activity suggesting
the existence of additional isoforms of a-DOX in pea
plants. Future work will serve to investigate the
occurrence of further a-DOX isoforms in pea and
the impact of the a-oxidation process and its
reaction products on the germination process.
Acknowledgments
We are grateful to Prof. H.-J. Gross, Prof. H.
Beier and Prof. U. Fischer for providing laboratory
space and facilities. We thank G. Grimmer for
expert technical assistance and kind support.
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