Physcomitrella patens has lipoxygenases for both eicosanoid and octadecanoid pathways

Article abstract of DOI:10.1016/j.phytochem.2008.11.012

Mosses have substantial amounts of long chain C20 polyunsaturated fatty acids, such as arachidonic and eicosapentaenoic acid, in addition to the shorter chain C18 α-linolenic and linoleic acids, which are typical substrates of lipoxygenases in flowering p

Full text of DOI:10.1016/j.phytochem.2008.11.012

Phytochemistry 70 (2009) 40–52
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Physcomitrella patens has lipoxygenases for both eicosanoid and
octadecanoid pathways
b
b
c
b
c
Aldwin Anterola a, , Cornelia Göbel , Ellen Hornung , George Sellhorn , Ivo Feussner , Howard Grimes
*
a Southern Illinois University, Carbondale, IL 62901-6509, USA
b University of Göttingen, D-37077 Göttingen, Germany
c Washington State University, Pullman, WA 99164-4234, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 11 July 2008
Received in revised form 30 October 2008
Available online 7 January 2009
Mosses have substantial amounts of long chain C20 polyunsaturated fatty acids, such as arachidonic and
eicosapentaenoic acid, in addition to the shorter chain C18 -linolenic and linoleic acids, which are typ-
ical substrates of lipoxygenases in flowering plants. To identify the fatty acid substrates used by moss lip-
a
oxygenases, eight lipoxygenase genes from Physcomitrella patens were heterologously expressed in
Escherichia coli, and then analyzed for lipoxygenase activity using linoleic,
acids as substrates. Among the eight moss lipoxygenases, only seven were found to be enzymatically
active in vitro, two of which selectively used arachidonic acid as the substrate, while the other five pre-
ferred a-linolenic acid. Based on enzyme assays using a Clark-type oxygen electrode, all of the active lip-
oxygenases had an optimum pH at 7.0, except for one with highest activity at pH 5.0. HPLC analyses
indicated that the two arachidonic acid lipoxygenases form (12S)-hydroperoxy eicosatetraenoic acid as
the main product, while the other five lipoxygenases produce mainly (13S)-hydroperoxy octadecatrienoic
a-linolenic and arachidonic
Keywords:
Physcomitrella patens
Funariaceae
Lipoxygenase
Dioxygenase
Lipid peroxidation
Oxylipin formation
pH dependent positional specificity
Substrate orientation
Eicosanoid
acid from
a
-linolenic acid. These results suggest that mosses may have both C20 and C18 based oxylipin
pathways.
Published by Elsevier Ltd.
1. Introduction
of different types (isoforms) of lipoxygenases in mammals. Forma-
tion of these eicosanoids is intriguingly similar to the formation of
Lipoxygenases are non-heme iron containing dioxygenases in-
volved in generation of lipid-derived signaling molecules in eukary-
otes. They catalyze the addition of molecular oxygen to fatty acids
containing a (1Z,4Z)-pentadiene functionality (e.g., arachidonic 1
plant oxylipins from a-linolenic (2) and linoleic (3) acids, suggesting
a possible generalized role of fatty acid oxygenation in defense sig-
naling (see Schemes 1 and 2 for chemical structures).
In flowering plants, linoleic (3) and a-linolenic (2) acids are the
and
a
-linolenic 2 acids), generating (1S,2E,4Z)-hydroperoxy fatty
most abundant fatty acids in lipid membranes and presumably serve
as the major substrates of lipoxygenases, which insert oxygen either
at the 9- or 13-positions of linole(n)ic acid, forming 9- and 13-hydro-
peroxy linole(n)ic acids, respectively. The 9-hydroperoxides formed,
namely 9-hydroperoxy octadecadienoic acid (9-HPODE, 4) and 9-
hydroperoxy octadecatrienoic acid (9-HPOTE, 5), are precursors of
a number of oxylipins, including: C9 aldehydes and oxoacids (found
in fruit flavors and scents) formed via the action of hydroperoxide
lyase (Mita et al., 2007); ketols and 10-oxo-11,15-phytodienoic acid
derived from 9,10-epoxy octadecatrienoic acid produced by a 9-spe-
cific allene oxide synthase (Hamberg, 2000; Itoh et al., 2002; Stumpe
et al., 2006); and colnele(n)ic acid generated by divinyl ether syn-
thase (Itoh and Howe, 2001; Fammartino et al., 2007; Stumpe
et al., 2001). 13-Hydroperoxy octadecatrienoic acid (13-HPOTE, 6)
acids, whichin turnserve as precursors to a variety of biologicallyac-
tive metabolites. Initially described as a lipoxidase in soybean (Balls
et al., 1943), this class of enzyme decades later gained considerable
attention in the biomedical community because of its involvement
in the inflammatory response (Borgeat et al., 1976). Indeed, several
inflammatory signaling molecules such as hydroxy eicosatetraenoic
acids (HETEs), leukotrienes, lipoxins and hepoxilins (Brenner and
Krakauer, 2003) are formed from arachidonic acid (1) via the action
Abbreviations: H(P)ETE, hydro(pero)xy eicosaetraenoic acid; 13-H(P)ODE, (13
S,9Z,11E)-13-hydro(pero)xy-9,11-octadecadienoic acid; 9-H(P)ODE, (9S,10E,12Z)-9-
hydro(pero)xy-10,12-octadecadienoic acid; 13-H(P)OTE, (13S,9Z,11E,15Z)-13-
hydro(pero)xy-9,11,15-octadecatrienoic acid; 9-H(P)OTE, (9S,10E,12Z,15Z)-9-
hydro(pero)xy-10,12,15-octadecatrienoic acid; KODE, keto octadecadienoic acid;
KOTE, keto octadecatrienoic acid; KETE, keto eicosatetraenoic acid (a.k.a. keto
arachidonic acid).
formed from a-linolenic acid (2) is similarly converted to another
set of oxylipins. The latter includes C6 aldehyde hexenal and traum-
atin formed by hydroperoxide lyase (Matsui, 2006), etherolenic acid
formed by divinyl ether synthase (Stumpe et al., 2008), and jasmonic
* Corresponding author. Tel.: +1 618 453 3222; fax: +1 618 453 3441.
E-mail address: anterola@siu.edu (A. Anterola).
0031-9422/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.phytochem.2008.11.012

A. Anterola et al. / Phytochemistry 70 (2009) 40–52
41
Scheme 1. Chemical structures of selected fatty acids and oxylipins.
Scheme 2. Chemical structures of selected oxylipins.
acid formed via a series of steps initiated by allene oxide synthase
(Wasternack, 2007). 9- and 13-Hydroperoxides are also converted
by peroxygenase (Blee, 1998) and epoxy alcohol synthase(Hamberg,
1999) to yield different epoxy alcohols.
Oxylipins have been implicated in defense and signaling (Blee,
2002; Rosahl and Feussner, 2005), although lipoxygenases them-
selves have also been proposed to be involved in seed germination
(via lipid mobilization with lipoxygenases acting on triglycerides)
(Feussner et al., 2001) and in nitrogen storage (with lipoxygenases
serving as vegetative storage proteins) (Fuller et al., 2001). Inter-
estingly, although flowering plants do not contain polyunsaturated
fatty acids such as arachidonic (1) and eicosapentaenoic acids, lip-
oxygenases (e.g., from soybean, barley, and potato) have been
found to use arachidonic acid (1) as substrate (Vörös et al., 1998;
Shimizu et al., 1984; Cook and Lands, 1975; Bild et al., 1977,
1978). It has been hypothesized that arachidonic (1) and eicosa-
pentaenoic acid from oomycete (such as Phytophtora infestans)
are utilized by plant lipoxygenases during pathogen attack to form
defense-related compounds (Schewe and Nigam, 1997).
Unlike flowering plants, algae and mosses may have substantial
levels of arachidonic (1) and/or eicosapentaenoic acids, which can
serve as substrates for their endogenous lipoxygenases. For exam-
ple, the red algae Rhodymenia pertussa (Jiang et al., 2000), Polyneura
latissima (Jiang and Gerwick, 1997), and Gracilariopsis lemaneifor-

42
A. Anterola et al. / Phytochemistry 70 (2009) 40–52
mis (Gerwick et al., 1991) have been reported to have oxylipins
and/or enzyme activities that suggest the existence of arachido-
nate/eicosapentaenoate 5-, 9- and 12-lipoxygenases, whereas
Chondrus crispus (another red alga) uses both arachidonic (1) and
linole(n)ic (2, 3) acids to produce predominantly 12-hydroperoxy
eicosatetraenoic acid (12-HPETE, 7) and 13-HPOTE (6)/13-HPODE
(8), respectively (Bouarab et al., 2004). Brown algae from the Lam-
inaria genus also use both C20 and C18 fatty acids, but apparently
profiles of seven heterologously expressed lipoxygenases in P. pat-
ens, which suggest that both C20 and C18 fatty acid based signaling
pathways may be present in this moss.
2. Results
2.1. Identification and sequence analysis of Physcomitrella
lipoxygenase genes
only those belonging to the
x6-group such as arachidonic (1) and
linoleic (3) acids, to form 15-HPETE (9) and 13-HPODE (8), respec-
tively (Gerwick, 1994). In the green algae Chlorella pyrenoidosa
(Nunez et al., 2002), a lipoxygenase active against linoleic acid
(3) has been described, which produces roughly equal amounts
of 9- and 13-HPODE (4 and 8). Hence both the eicosanoid and octa-
decanoid pathways have been apparently found in diverse forms of
algae. Cyanobacteria have also been shown to harbor 13-lipoxy-
genases in the case of Nostoc sp. (Lang and Feussner, 2007; Lang
et al., 2008), and 9/13-lipoxygenases in the case of Oscillatoria sp.
(Beneytout et al., 1989).
In mosses, only a single lipoxygenase, PpLOX1 from P. patens,
has so far been identified and characterized (Senger et al., 2005).
It is mainly an arachidonate 12-lipoxygenase (producing 12-
HPETE, 7) with 13-lipoxygenase activity against linole(n)ic acid
(2, 3). Although the observed activity of this single enzyme ac-
counted for oxylipins previously detected in P. patens (Wichard
et al., 2004), this does not preclude the existence of other lipoxyge-
nase isoforms in this moss. Indeed, in most plant species, lipoxy-
genases typically exist as members of multi-gene families
(reviewed in Feussner and Wasternack, 2002), whose encoded pro-
teins may have different biochemical properties. Soybean, for
example, has eight isoforms known, of which three are expressed
in seeds (Axelrod et al., 1981) and five are in vegetative tissues
(e.g., leaves) (Fuller et al., 2001). These isoforms differ in terms of
their substrate specificities, pH optima and the relative proportion
of products formed (Fuller et al., 2001). Added to the fact that lip-
oxygenases have been found in various subcellular locations
including the mitochondria (Braidot et al., 2004), cytosol, vacuole,
chloroplast and the plasma membrane (reviewed in Feussner and
Wasternack, 2002), such differences may reflect the variety of
physiological functions lipoxygenases perform in plants, most of
which are not yet fully understood.
Twelve Physcomitrella putative transcripts (i.e., expressed se-
quence tags or ESTs) in Physcobase (http://moss.nibb.ac.jp/), a
DNA database maintained by the National Institute of Basic Biology
(NIBB) in Okazaki, Japan (Nishiyama et al., 2003), were found to be
highly similar (>40%) to soybean lipoxygenases via a BLAST search
using soybean lipoxygenase-1 as a query sequence. EST clones that
had the longest 50 region were identified from the database, and
then sequenced to obtain full-length nucleotide information,
which indicated that only eight of them (pphn43n22, pphb33n05,
pphb15j12, pphb13L22, pphb14h18, pphn9o24, pphn50o06, and
pph20m14) encoded ꢀ100 kDa lipoxygenases. The other four
cDNAs are most likely ‘‘processed pseudogenes” which though
transcribed into mRNAs are not translated into functional proteins
(Vanin, 1985). Indeed, these pseudogenes contain only part of the
lipoxygenase sequence which would have predicted translational
products of only 30–65 kDa, whereas all known naturally occurring
lipoxygenases from flowering plants are ꢀ100 kDa.
Among the remaining eight putative LOX genes, one of them
(pph20m14) did not yield an active LOX protein under the expres-
sion and assay conditions used. Hence, only seven had been func-
tionally proven to be active LOXs in vitro (Table 1), with one of
them (pphb33n05) being essentially identical (99.6% at the amino
acid level) to the previously reported Physcomitrella lipoxygenase
PpLOX1 (Senger et al., 2005). Their 50 and 30 untranslated regions
were nearly identical, except for the longer cDNA sequence of
pphb33no05. The latter EST also had an additional codon (GAG)
in position 1378 of the coding region of the cDNA, which added
a glutamate in the translated protein. The other two differences
in the coding region involved adenine to guanine base changes
in positions 658 and 2496 in the pphb33n05 sequence, which
changed a glutamate to a glycine in the N-terminal region and a
lysine to a glutamate in the C-terminal region of the protein,
respectively. Hence, this EST represents either a more complete
sequence of the same gene than originally reported, a splice var-
iant, or a distinct gene altogether. To distinguish this EST from the
original sequence, while at the same recognizing that they may be
the same gene, this EST is thus referred to in this paper as
PpLOX1b.
Mosses are especially interesting to study because they are phy-
logenetically distinct from seed plants, and are hypothesized to
have evolved separately from algal predecessors (Graham et al.,
2000). While extant marine algae are known to have polyunsatu-
rated fatty acids and even produce mammalian-like eicosanoids
(Gerwick and Bernart, 1993), flowering plants appear to have lost
this metabolic capacity in the course of evolution. Mosses, along
with other non-flowering plants, have retained the ability to form
polyunsaturated fatty acids, but it is still not clear what types of li-
pid signaling pathways these earlier land plants have, e.g., what
fatty acids are used to form lipoxygenase-derived oxylipins. In this
contribution, we report the substrate preferences, product and pH
Following the earlier nomenclature, the six other active LOXs
were named PpLOX2 (pphn43n22), PpLOX3 (pphb15j12), PpLOX4
(pphb13L22), PpLOX5 (pphb14h18), PpLOX6 (pphn9o24) and
PpLOX7 (pphn50o06). The seven PpLOXs encode 105, 105, 103,
104, 108, 106 and 109 kDa proteins, respectively. The last EST
Table 1
Structural and functional characteristics of lipoxygenase genes in Physcomitrella patens.
Name
Accession
no.
50-UTR
(bases)
30-UTR
(bases)
Coding region
(bases)
Predicted protein size
(kDa)
Preferred fatty acid
substrate
Activity on preferred
substrate
PpLOX1b
PpLOX2
PpLOX3
PpLOX4
PpLOX5
PpLOX6
PpLOX7
ABF66647
ABF66648
ABF66649
ABF66650
ABF66651
ABF66652
ABF66653
479
174
245
397
216
217
199
259
229
359
218
252
294
138
2817
2817
2763
2763
2856
2871
2901
105
105
103
104
108
106
109
Arachidonate
Arachidonate
(12S)-LOX
(12S)-LOX
(13S)-LOX
(13S)-LOX
(13S)-LOX
(13S)-LOX
(13S)-LOX
a
a
a
a
a
-Linolenate
-Linolenate
-Linolenate
-Linolenate
-Linolenate

A. Anterola et al. / Phytochemistry 70 (2009) 40–52
43
(pph20m14) had a calculated protein molecular weight of 94 kDa.
The putative 94 kDa protein had only 828 aa compared to at least
920 in other PpLOXs, with less amino acids in the N-terminal.
While the C-terminal appears to be the same length as the other
PpLOXs, the C-terminal isoleucine residue found conserved in
other plant LOXs is missing (Fig. 1).
Among the active PpLOX genes, PpLOX1b and 2 are most closely
related to each other, being 96.1% identical at the amino acid level.
Fig. 1. Amino acid sequences of active lipoxygenases of P. patens (PpLOX1b to 7) aligned using Clustal W. Conserved histidines, asparagines and isoleucines are italicized and
marked with arrows.

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A. Anterola et al. / Phytochemistry 70 (2009) 40–52
LOX1:St:11
LOX1:St:12
LOX1:St:10
LOX1:St:1
LOX1:St:5
LOX1:St:8
LOX1:St:9
LOX1:St:4
LOX1:St:3
LOX1:St:6
LOX1:Le:1
LOX1:St:7
LOX1:Le:2
LOX1:Le:3
LOX1:Nt:1
LOX1:At:2
LOX1:St:2
LOX1:At:1
LOX2:Le:2
LOX2:St:2
LOX2:At:3
LOX2:At:2
LOX2:At:4
LOX2:At:1
LOX2:Le:1
LOX2:St:1
PpLOX7
92
93
97
55
76
100
77
62
100
100
100
99
82
97
96
96
100
100
100
100
91
61
100
100
96
100
PpLOX8
PpLOX1
100
100
PpLOX2
PpLOX3
95
PpLOX4
PpLOX5
100
78
PpLOX9
64
PpLOX6
Fig. 2. Phylogenetic tree of P. patens lipoxygenases (PpLOX1 to 7) together with selected lipoxygenases from angiosperms (sequences taken from Feussner and Wasternack,
2002). PpLOX8 (EDQ50470) and PpLOX9 (EDQ67347) were from EMBL. The evolutionary history was inferred using the Neighbor-Joining method. The percentages of
replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) are shown next to the branches.
In the ESTs we sequenced, PpLOX1b cDNA was 3594 nucleotides
long, with 259 bases in the 30-UTR (untranslated region) followed
by a polyA tail, and 479 bases comprising the 50-UTR, which is sig-
nificantly longer than in the previously isolated PpLOX1 mRNA
(only 82 bases). With this new sequence information, we were able
to confirm the previous assumption of the ATG translational start
site for the PpLOX1 mRNA, since the two upstream ATGs in the
same reading frame were both followed by a stop codon. For
PpLOX2, a 3256-nucleotide cDNA sequence has been determined
from the EST clone pphn43n22, having a relatively shorter 50-
UTR (174 bases) which did not show any stop codons prior to
the first putative ATG start site. However, at the time of writing,
a new EST (pph18f10) has been deposited in Physcobase which
showed stop codons in the same reading frame prior to this pre-
sumed start codon, thus increasing the likelihood that we have ob-
tained the full-length coding region of this gene. Additionally,
PpLOX2 encodes a similar-sized protein (105 kDa) with same num-
ber of amino acids (938) as the new PpLOX1b sequence, hence it is
very likely that we have the complete sequence for the encoded
protein. Interestingly, PpLOX2 also has the additional ‘‘GAG” in
the coding region, which inserts a Glu in the translated protein.
Thus both PpLOX2 and the new PpLOX1b sequence have 938 ami-

A. Anterola et al. / Phytochemistry 70 (2009) 40–52
45
no acids, while the previously reported PpLOX1 has only 937.
Nonetheless, the presence of this additional residue did not seem
to affect LOX activity. Using the TargetP v1.0 algorithm (Emanuels-
son et al., 2000), putative transit peptides were found in each of
these protein sequences which possibly direct them to the chloro-
plast. They are therefore classified as type 2 lipoxygenases (Feuss-
ner and Wasternack, 2002).
bases in the 50-UTR, coding region and 30-UTR, respectively. In each
case, the putative start site is preceded by a stop codon in the same
reading frame in the 50-UTR, which supports a predicted transla-
tional product of 920 amino acids for both cDNAs, each of which
is only 40% identical to either PpLOX1 or PpLOX2. Neither PpLOX3
nor PpLOX4 has a putative N-terminal signal peptide, according to
TargetP v1.0 prediction.
PpLOX3 and 4, which share 73.4% amino acid identity, are more
closely related to each other than to the rest of the PpLOX family.
PpLOX3 cDNA from the EST clone pphb15j12 is found to be 3383
nucleotides long, which includes 245 bases in the 50-UTR, 2763
in the coding region, and 359 in the 30-UTR followed by a polyA tail.
PpLOX4 cDNA is 3409 nucleotides long, with 397, 2763 and 218
PpLOX5, 6 and 7 have the longest ORFs among the active
PpLOXs, encoding a stretch of 951, 956, and 966 amino acids,
respectively. They are also most divergent from each other and
from the other active PpLOXs, with only 42–47% identity at
the amino acid level. PpLOX5 cDNA has 216 bases in the 50-
UTR, a 2856-nucleotide coding region and a 30-UTR with 252
Fig. 3. The enzymatic activities at various pHs of P. patens lipoxygenases PpLOX1b (A), PpLOX2 (B), PpLOX3 (C), PpLOX4 (D), PpLOX5 (E), PpLOX6 (F) and PpLOX7 (G), using
arachidonic (1) (s, d), -linolenic (2) (N, ) and linoleic (3) (j, h) acids as substrates. Open symbols: phosphate buffer; shaded symbols: citrate buffer.
a
D

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A. Anterola et al. / Phytochemistry 70 (2009) 40–52
bases followed by a polyA tail, totaling 3340 bases. PpLOX6, on
the other hand, has 3398 nucleotides comprising the 217,
2871, 294 and 16 bases in the 50-UTR, ORF, 30UTR and polyA tail,
respectively. PpLOX7 has a relatively short 50-UTR (199 bases),
the longest ORF (2901 bases) among the PpLOXs, and the short-
est 30-UTR (138 bases which include 16 adenines in the polyA
tail). Based on the prediction results of TargetP v1.0, PpLOX5 is
possibly mitochondrial, whereas PpLOX6 and PpLOX7 are
plastidic.
All six lipoxygenase proteins (PpLOX2–PpLOX7) described
above harbor the conserved histidine, asparagine and isoleucine
residues involved in iron binding (Fig. 1). However, as described
for PpLOX1 (Senger et al., 2005), none of them harbored the deter-
minants involved in positional specificity described for lipoxygen-
ases from flowering plants. Phylogenetic analysis with selected
lipoxygenases from flowering plants showed that PpLOX7 group
together with type 2 lipoxygenases from flowering plants, while
the other PpLOXs form a distinct clade (Fig. 2).
2.2. Substrate specificity and pH profiles of PpLOXs
The open reading frames of PpLOX1b to 7 and EST clone
pph20m14, were heterologously expressed in Escherichia coli using
pET101 as vector and BL21DE3 as host strain. After induction with
IPTG at 20 °C for 24 h, bacterial lysates were assayed for LOX activ-
ity at different pHs, using linoleic (3),
a-linolenic (2) and arachi-
donic (1) acid as substrates, respectively. Using the Clark-type
oxygen electrode, only PpLOX1b to 7, and not pph20m14, were
found to be active, each with varying substrate preferences and
pH optima.
Consistent with their high sequence similarity, PpLOX1b and 2
had the same pH profile and substrate preference (Fig. 3A and B).
Both proteins have pH optima at pH 7.0 and a preference for ara-
chidonic acid (1), which agrees with previous findings on PpLOX1
(Senger et al., 2005). Both PpLOX1b and PpLOX2 are most active
against arachidonic acid at a relatively narrow pH range from pH
6.5 to 7.5, with a gradual decrease in LOX activity as the pH is de-
Fig. 4. HPLC analyses of LOX reaction products. PpLOX1b with arachidonic acid (1) (A), PpLOX7 with arachidonic acid (1) (B) and with a-linolenic acid (2) (C). The insets show
the respective enantiomer separation of the products.

A. Anterola et al. / Phytochemistry 70 (2009) 40–52
47
Fig. 5. Relative amounts of products generated by P. patens lipoxygenases as analyzed and quantified by normal phase HPLC, using linoleic (3),
(2) acids as substrates.
a
-linolenic (2) and arachidonic
creased from pH 6.0 to 4.0. A sudden drop in the activity is seen at
pH 8.0 and even decreases further as the pH is shifted to increased
alkalinity. Under the current assay conditions used, no significant
tially utilizes both
strates, whereas PpLOX4 clearly prefers
Nevertheless, closer inspection of the pH profiles of PpLOX3 and
PpLOX4 activities against -linolenic acid (2) shows a very similar
a
-linolenic (2) and arachidonic (1) acids as sub-
a
-linolenic acid (2) only.
activity has been observed when either linoleic (3) or
a
-linolenic
a
(2) acid is used as substrate. Although our data show that PpLOX2
is more active than PpLOX1b, this may simply be due to differences
in expression levels. The latter, however, cannot be determined at
this point, because the heterologously expressed proteins cannot
be distinguished from endogenous bacterial proteins in SDS–PAGE
gels (data not shown). The expression levels can therefore still be
optimized prior to our next step of purifying these proteins.
PpLOX3 and 4, while similar in their primary structure, have
distinct substrate preferences (Fig. 3C and D). PpLOX3 preferen-
trend. In each case, gradual decrease in activity was observed as
the pH is adjusted away from their respective pH optima (6.5 for
PpLOX3 and 7.0 for PpLOX4), with a discernible shoulder in the
alkaline region (pH 9.5 and pH 9.0, respectively) reflecting a slight
increase in activity.
The activity of PpLOX3 against arachidonic acid (1) is highest at
pH 6.5 to 8.0, being relatively constant at this pH range, which then
gradually decreases as the pH is changed in either direction. With
a-linolenic acid (2), PpLOX3 activity is highest at pH 7.0, showing a

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A. Anterola et al. / Phytochemistry 70 (2009) 40–52
gradual decline as the pH is increased to pH 10.0, until activity is
no longer detected at pH 10.5. Following a drop in activity at pH
6.5 from pH 7.0, a gradual decrease in activity is observed from
pH 6.5 to 4.0, until the enzyme is no longer active at pH 3.5.
PpLOX4 is slightly active against arachidonic acid (1) with high-
est activity observed at pH 7.0, though the activities at pH 7.0 and
8.0 are almost up to the same level. In fact, PpLOX4 activity against
arachidonic acid (1) did not decrease significantly until the pH is
either below pH 6.0 or at pH 8.5 and higher, with negligible activity
detected at pH 3.5 and 9.5. This is somewhat similar to the pH
profile of PpLOX4 activity with linoleic acid (3) as substrate,
except that the enzyme remains active at pH 9.0–10.0, but not at
pH 10.5.
cedure which utilized reversed, normal and chiral phase HPLC
(Senger et al., 2005), confirmed that both PpLOX1b and PpLOX2
were arachidonate (12S)-LOXs, similar to what was previously
determined for PpLOX1 (Senger et al., 2005). We found no evidence
of changes in the product specificity of either PpLOX1b or 2 that
accompanied alteration in the pH of the reaction.
We achieved baseline separation of the LOX products of a-lino-
lenic acid (2), i.e., 9-HOTE (16) and 13-HOTE (17), by reversed
phase HPLC (data not shown), and thus were able to determine
that PpLOX3, 4, 5, 6 and 7 are all 13-LOXs with respect to a-linole-
nic acid (2), and 15-LOXs with respect to arachidonic acid (1). This
was confirmed by normal phase HPLC, which additionally was able
to determine that 13-HODE (18) was formed as the major product
from linoleic acid (instead of 9-HODE, 19). Representative chro-
matograms of LOX products using normal phase and chiral HPLC
are shown in Fig. 4.
PpLOX5 prefers
has significant, albeit lower, activity with arachidonic acid (1), and
much less activity against linoleic acid (3) (Fig. 3E). With -linole-
a-linolenic acid (2) as substrate, although it also
a
nic acid (2), the pH optimum appears to be at pH 7.0, although al-
most the same level of activities have been observed at pH 5.5 and
6.0, following a significant drop at pH 6.5. A steep decline in activ-
ity then ensues with further increase in pH from 7.0 or a decrease
from pH 5.5. With arachidonic acid (1), the pH optimum of PpLOX5
is at pH 6.5, with gradual decrease in activity as the pH decreases
to 4.0. A steeper decline is observed from pH 7.0–8.0, with the en-
zyme no longer active at pH 8.5. Much less activity was observed
with linoleic acid (3) as substrate, though activity stayed constant
at a broader pH range (5.5–7.0), with gradual decrease as the pH is
decreased from 5.5 to 4.0 or when the pH is changed from 7.0 to
8.0. Linoleic acid (3)-dependent LOX activity of PpLOX5 may be
considered negligible when compared to the activity against lino-
lenic acid (2). PpLOX5 activities remained low at pH 8.5 and 9.0
using any of the three substrates.
Fig. 5 shows each of the PpLOX’s product profiles, which were
obtained by normal phase HPLC. PpLOXs are essentially indistin-
guishable from each other with respect to their activities against
linoleic acid (3). PpLOX4 generated the highest proportion of 13-
HODE (18) (ꢀ80%), followed by PpLOX5 (ꢀ67%) and PpLOX3
(ꢀ63%). PpLOX7 had the lowest proportion of 13-HODE (18)
(ꢀ54%), whereas PpLOX6 had about the same proportion of 13-
HODE (18) as that of PpLOX2 (58%). In each case, the remaining
products were mostly 9-HODE (19) (17–38%), with minimal
amounts of 13-KODE (20) (ꢀ0.7–5%) and 9-KODE (21) (ꢀ0.4–2%)
detected. The similarity in their product profiles is not surprising
given that none of them actually prefers linoleic acid (3) as sub-
strate (see Schemes 1 and 2 for chemical structures).
With
a-linolenic acid (2) as substrate, the order of PpLOXs in
terms of the highest proportion of 13-HOTE (17) produced is differ-
ent from that seen with using linoleic acid (3). Excluding PpLOX1b
and PpLOX2 from the comparison, PpLOX4 and 5 had the lowest
proportion of 13-HOTE (17) produced (ꢀ51 and 62%, respectively)
whereas PpLOX6 had the highest (ꢀ93%). PpLOX1b and 2 can be
PpLOX6 is most active under acidic conditions, with only mini-
mal activity observed at pH 7.0 and higher (Fig. 3F).
a-Linolenic
acid (2) is the preferred substrate, being oxygenated 5-fold more
than arachidonic acid (1), and 10-fold more than linoleic acid (3)
at the optimal pH range (4.5–5.0). While the enzyme is still active
against the preferred substrate even at pH 3.5, a steep decline in its
excluded from this comparison since neither prefers
a-linolenic
acid (2) as substrate. Indeed, although PpLOX1b and 2 seemed to
form 12-HOTE (22) as the major product, the absolute amounts
of these hydroxy fatty acids were so low compared to those formed
by PpLOX1b and 2 from arachidonic acid (1) (see below), that they
could simply be derived from non-specific oxidation.
The product profiles of the PpLOXs with arachidonic acid (1)
were most instructive for comparative purposes since all PpLOXs
used this as substrate. Thus, PpLOX1b and 2 formed mostly12-
HETE (12) (56% and 79%, respectively) with minor amounts of
15-HETE (10) (ꢀ10–20%), 5-HETE (13) (ꢀ4–7%), 8-HETE (14)
(ꢀ3–5%), and 11-HETE (11) (ꢀ3–7%), whereas PpLOX3, 4, 5, 6
and 7 formed 15-HETE (10) as the major product (ꢀ74%, 69%,
78%, 67% and 82%, respectively). These results show that PpLOX1b
and 2 are arachidonate 12-LOXs, while the rest of the PpLOXs are
arachidonate 15-LOXs, which is consistent with their respective
activity against a-linolenic acid (2) is observed from pH 4.5 to 3.0,
whereas the decrease in activity is more gradual when the pH is in-
creased from 5.0 to 6.0. A sudden drop in activity is observed at pH
6.5 decreasing further at 7.0, where activity holds at a minimal le-
vel until pH 8.0. In the case of arachidonic (1) and linoleic (3) acids,
the decrease in activity as the pH is adjusted from 4.5 to 5.0,
respectively, is more gradual, as the activities with these substrates
are already relatively low compared to that with
a-linolenic acid.
PpLOX7 prefers -linolenic (2) over either linoleic (3) or arachi-
a
donic (1) acids, with optimal activity at pH 7.0 (Fig. 3G). A steep de-
cline in activity is observed as the pH is adjusted towards either
acidic or alkaline conditions, although a considerable level of activ-
ity still persists around pH 8 before PpLOX becomes completely
inactive at pH 9. It is interesting to note that none of the moss lip-
oxygenases exhibited optimal activity at alkaline pH.
activities with a-linolenic acid (2) as a 13-LOX.
Analysis of the enantiomers of LOX products formed by each
PpLOX was carried out by chiral phase HPLC analysis, with the re-
sults shown graphically in Fig. 6. With linoleic acid (3) as sub-
strate, PpLOX1b formed racemic mixtures of both 13-HODE (18)
and 9-HODE (19), which is consistent with its lack of significant
activity using this substrate. Hence, the LOX products formed
from PpLOX1b incubation may simply be a result of non-specific
oxidations. In the case of PpLOX3, 4, 5, 6 and 7, the S-enantiomer
of 13-HODE (18a) is mostly formed (86%, 88%, 78%, 86%, 78%,
respectively), whereas a racemic mixture of 9-HODE (19) was
2.3. LOX product analysis
The products formed by each PpLOX at different pHs (4.0–8.5)
using the preferred substrates arachidonic (1) and
a-linolenic (2)
acids were initially analyzed by reversed phase HPLC. The elution
conditions developed herein for the reversed phase HPLC analysis
of expected LOX products from arachidonic acid (1) (see Section
5) allowed the separation of 15-HETE (10), 11-HETE (11), 12-HETE
(12) and 5-HETE (13), although 8-HETE (14) and 9-HETE (15) coe-
luted with 12-HETE (12) and 11-HETE (11), respectively (data not
shown). Under these separation conditions, PpLOX1b and PpLOX2
accumulated a major peak that coeluted with 8-HETE (14) and
12-HETE (12). Further HPLC analysis of the LOX products via a pro-
produced. Similar results were obtained with
a-linolenic acid
(2) as substrate. PpLOX1b generated a racemic mixture of either
13-HOTE (17) or 9-HOTE (16), whereas the other PpLOXs formed
almost enantiomerically pure (13S)-HOTE (23). PpLOX3, 6 and 7

A. Anterola et al. / Phytochemistry 70 (2009) 40–52
49
Fig. 6. Enantiospecificity of P. patens lipoxygenases with respect to the products generated from linoleic (3),
a
-linolenic (2) and arachidonic (1) acids. Proportions of the
different isomers are given in Fig. 5.
generated almost exclusively the S-enantiomer of 13-HOTE (23)
(96%, 98% and 94% e.e., respectively), whereas PpLOX4 and 5
had high proportions of (13S)-HOTE (23) as well (96% and 90%,
respectively). This higher enantiomeric purity of the 13-HOTE
(17) products (compared to 13-HODE, 18) probably reflects their
preference for linolenic acid (3) as substrate. Interestingly,
PpLOX2 forms an excess of the S-enantiomers of 13-HODE (18a)
and 13-HOTE (17) (36% and 66% e.e., respectively), instead of a
racemic mixture.
In the case of arachidonic acid (1), PpLOX1b and 2 generated
95–99% of the (12S)-HETE (24) enantiomer, while the other prod-
ucts, 5-HETE (13), 11-HETE (11), 8-HETE (14) and 15-HETE (10),
were racemic. The rest of the PpLOXs formed racemic products
from arachidonic acid (1) except for 15-HETE (10), in which the
S-enantiomer (25) is generated preferentially (ꢀ79–94%), which
is again consistent with their 13-LOX activity against
a-linolenic
acid (2). Hence, all of the PpLOXs cloned thus far stereospecifically
generate LOX products of the S-configuration.

50
A. Anterola et al. / Phytochemistry 70 (2009) 40–52
chidonic (1) and linoleic (3) acids, so there is a strong possibility
3. Discussion
that they actually produce 13-HPOTE (6) in vivo. If this is the case,
then mosses may also be producing octadecanoids, in addition to
eicosanoids.
An optimal pH of 5.0 for PpLOX6 is compatible with the poten-
tial acidification of the thylakoid lumen (Lee and Kugrens, 1999), if
this is where PpLOX6 is ultimately targeted, although all type 2 lip-
oxygenases have been localized to the stroma so far. A lipoxyge-
nase from barley also had an acidic pH optimum at 6.0 (Lulai and
Baker, 1976), but this has not been specifically localized to any
organelle.
Full-length cDNAs from Physcobase facilitated the identification
and functional characterization of lipoxygenase genes in P. patens.
Since we no longer had to perform cDNA library screening and/or
PCR-based cloning, functional characterization of these genes had
been completed in less time and was also less prone to error. Fur-
thermore, since Physcobase had already assembled the ESTs into
putative unigenes, it is possible to verify the sequence and/or iden-
tify sequence variants by sequencing other ESTs belonging to one
contig pair. On the other hand, these ESTs do not represent the
complete genome of P. patens, and so there still is the possibility
of more lipoxygenase isoforms present in the moss genome. In-
deed, at least four more additional lipoxygenase genes may be
present in P. patens, based on its recently sequenced genome
(Rensing et al., 2008). However, none of these additional lipoxy-
genases reported in the moss genome are found in the EST dat-
abases. Given the limited number of Physcomitrella ESTs available
at present, it is difficult to determine whether or not these addi-
tional lipoxygenase-like sequences are expressed and encode ac-
tive lipoxygenases.
Interestingly, none of the Physcomitrella lipoxygenases are 9-
lipoxygenases with respect to
a-linolenic acid (2) nor are there
PpLOXs that prefer linoleic acid (3). Linoleic acid (3) is found
mostly in seeds of flowering plants where lipoxygenases are
thought to be important for germination. As early land plants do
not have seeds, the substrate preference of PpLOXs is consistent
with the possible evolution of linoleate-utilizing LOXs in seed
plants. Furthermore, it seems that the distribution of 9-lipoxygen-
ases in the plant kingdom may be restricted to flowering plants
and the existence of 9/13-lipoxygenases in angiosperms suggest
that the early forms of land plant lipoxygenases are 13-lipoxygen-
ases, which later evolved to also produce 9-lipoxygenase products.
It appears however that the ability to utilize arachidonic acid (1) is
an ancient trait since this is also present in algae. Alternatively, the
ability to use arachidonic acid (1) as a LOX substrate has evolved
independently in several lineages of the plant kingdom.
The functional characterization of lipoxygenases in the moss P.
patens, following the report of a multifunctional lipoxygenase from
the same species (Senger et al., 2005), sheds more light into the
nature of oxylipin metabolism in non-flowering land plants.
Mosses differ from higher plants in terms of their lipid composi-
tion, especially in having significant amounts of arachidonic acid
Phylogenetic analysis of moss lipoxygenases together with
other known plant lipoxygenases showed that all PpLOXs except
for PpLOX7 clustered together into one clade (Fig. 2). This suggests
that the ancestor of PpLOX7 gave rise to type 2 LOXs and perhaps
more recently to type 1 LOXs in higher plants, while the other
PpLOXs form a distinct clade that is probably bryophyte specific.
It is also possible that PpLOXs and lipoxygenases from higher
plants evolved from a common cyanobacterial ancestor that has
been described recently (Lang and Feussner, 2007; Lang et al.,
2008).
(1) comparable to that of linoleic (3) and
a-linolenic (2) acids
(Dembitsky, 1993). P. patens is in fact reported to have more ara-
chidonic (1) (16.1%) than either linoleic (3) (13.7%) or linolenic
(2) (11.9%) acids (Grimsley et al., 1981). Hence, P. patens and other
mosses could potentially have a C20-based lipoxygenase pathway
similar to mammalian counterparts, which seemed the only plau-
sible working hypothesis prior to our discovery that other lipoxy-
genases do exist in this moss that utilize
a-linolenic (2) more so
than arachidonic (1) acid. Linole(n)ic acid (2, 3) derived oxylipins
such as 9-HPODE (4), 13-HPODE (8), 9-HPOTE (5) and 13-HPOTE
(6) have previously been detected in P. patens (Stumpe et al.,
2006), but this could have also been derived non-enzymatically
or non-specifically via an arachidonate lipoxygenase. Now it is
clear that both C18 and C20 fatty acids could be sources of oxyli-
pins in P. patens since this moss has a variety of lipoxygenases with
distinct substrate preferences, including for C18 fatty acids. Since
the existence of both C18- and C20-derived oxylipins has already
been observed in algae (Moghaddam and Gerwick, 1990; Gerwick
et al., 1999; Jiang et al., 2000; Bouarab et al., 2004; Tsai et al.,
2008), it makes sense that mosses have the same types of oxylipins
produced, as they represent a distinct lineage phylogenetically in
between algae and higher plants.
4. Concluding remarks
Physcomitrella patens has several lipoxygenases with different
substrate and product specificities. Among the seven lipoxygenas-
es that have been functionally characterized, two are arachidonate
(12S)-lipoxygenases and the remaining five are linolenate (13S)-
lipoxygenases. These results are consistent with mosses having
both eicosanoid and octadecanoid pathways, similar to those found
in algae. This provisionally provides an evolutionary explanation
for the arachidonate utilizing lipoxygenase activities observed in
some angiosperms which do not produce arachidonic acid (1).
The results also suggest that other arachidonic acid (1) producing
plants (such as some gymnosperms and ferns), may have C20-de-
rived oxylipins as well. Further investigation of lipoxygenases in
these plants could thus result in more insights into the evolution
and diversity of oxylipin pathways.
There seems to be at least two Physcomitrella lipoxygenases act-
ing on arachidonic acid (1), namely PpLOX1b and PpLOX2, which
apparently have strong preference for this substrate, and produce
(12S)-HPETE (26) as the major product. PpLOX3 also appear to uti-
lize arachidonic acid (1) at apparently the same efficiency as a-lin-
olenic acid (2), but the product formed is (15S)-HPETE (27).
Furthermore, all three enzymes are predicted to be in the plastid.
If this prediction based on phylogenetic tree analysis is correct,
then (12S)-HPETE (26), as well as its lyase product 12-ODTE (28)
(Senger et al., 2005), would be expected to be generated in the
plastids. Whether or not LOX products generated in the plastids
are substrates for other enzymes remains to be determined.
In higher plants, 13-HPOTE (6) is the substrate of AOS and HPL
in the chloroplasts. 13-HPOTE (6) could preferentially be generated
by PpLOX3 to 7, all of which may be localized in the plastid.
5. Experimental
5.1. Materials
All solvents used were HPLC grade (Fisher). Arachidonic (1), lin-
olenic (2), linoleic (3) and hydroxyfatty acids (10–19) were pur-
chased from Cayman Chemical (Ann Arbor, MI). All other
reagents were analytical or molecular biology grade (from Sigma
unless otherwise noted). Physcomitrella cDNA clones (pphn50o06,
pphb14h18, pphb33n05, pphb14L11, pph20m05, pphn43n22,
PpLOX4, 5, 6 and 7 all appear to prefer a-linolenic acid (2) over ara-

A. Anterola et al. / Phytochemistry 70 (2009) 40–52
51
pphn35p15, pphn9o24, pphb15j12, pph17d11, pph20m14,
pphb13L22) were obtained from RIKEN BioResource Center
(Nishiyama et al., 2003).
(Hansatech Instruments, Norfolk, UK) by measuring the decrease
in oxygen levels in a 0.5 mL enzyme reaction mixture. The latter
consisted of either 0.1 M phosphate or citrate buffer (485
0.1–0.4 mg crude protein extract (i.e., 5–15 L bacterial lysate)
and 5 M arachidonic (1), -linolenic (2) or linoleic (3) acids.
lL),
l
5.2. cDNA sequencing and analysis
l
a
Lyophilized plasmid DNA (100 ng) harboring putative LOX
genes (from RIKEN BRC) were reconstituted in nuclease-free H2O
5.5. HPLC analysis of LOX products
(5
(Stratagene) via electroporation. Transformants were grown in
selective LB media containing ampicillin (100 g/ml) at 37 °C. Plas-
l
L) and transformed into electrocompetent XL1-Blue cells
Bacterial lysates (prepared from cell pellets as in Section 5.4,
L) were incubated with 10 M arachidonic (1), -linolenic
(2) or linoleic (3) acids (10 L) in 0.1 M phosphate buffer pH 7.0
(380 L) for 30 min. After addition of HOAc–H2O (0.5 mL, 3:97, v/
v), lipids were extracted using the Bligh and Dyer method (Bligh
and Dyer, 1959) and the CHCl3 extract was dried under reduced
pressure using a centrifugal evaporator (Jouan RC10-22, Winches-
ter, VA). Hydroperoxyfatty acids were reduced to the correspond-
ing hydroxyfatty acids by addition of NaBH4 (100 mM) in MeOH
10
l
l
a
l
l
mids were prepared from overnight-grown positive colonies after
growing in 5 mL LB media containing ampicillin (100 g/mL) at
37 °C for 16 h, using a Promega WizardÒ plus SV kit, which were
then used as template for a DNA sequencing reaction containing
BigDye reagent and T7 or T3 primer. PCR consisted of a 96 °C hot
start for 3 min followed by 25 cycles of 96 °C/20 s denaturation,
50 °C/30 s annealing, and 60 °C/3 min elongation performed on
an Amplitron II thermocycler. PCR products were purified by pass-
ing them through PerformaÒ gel filtration cartridges (Edge BioSys-
tems), and analyzed using an ABI sequencer. Subsequent DNA
sequencing reactions were performed using internal primers de-
signed from the initial sequences obtained to determine the full-
length sequences of the cDNAs, which were eventually assembled
from the overlapping sequence data generated from these
sequencing reactions. Sequence information was processed and
analyzed using GCG software (University of Wisconsin, Madison,
WI).
l
(50
prior to HPLC analysis of a 10
To identify LOX products from arachidonic (1) and
l
L). After 30 min HOAc–H2O (50
lL, 0.5:99.5, v/v) was added
lL aliquot.
a
-linolenic
TM
(2) acids, reversed phase HPLC was performed on a Waters 626
LC system connected to a 996 Photodiode Array Detector and a
717 plus autosampler, using
a
Novapak C18 column
(3.9 ꢂ 150 mm, 4
l
m particle size). For analysis of arachidonic acid
(1) derived products, samples were eluted with a linear gradient
from MeCN–H2O–HOAc (30:69.65:0.35) to MeCN–H2O–HOAc
(50:49.75:0.25) in 10 min, followed by isocratic elution with
MeCN–H2O (50:50) for 30 min. LOX products from a-linolenic acid
(2) were separated via isocratic elution with MeCN–H2O–HOAc
(50:59.97:0.03). Hydroxyfatty acids derived from C18 and C20
fatty acids were detected at 234 and 237 nm, respectively. Lipoxy-
genase products were also analyzed independently by normal
phase HPLC followed by chiral phase HPLC to determine their
enantiomeric forms. LOX products were identified by comparison
of their retention times and absorption spectra with authentic
standards purchased from Cayman Chemical (Ann Arbor, MI) or
generated in the the laboratory, as described (Senger et al., 2005).
5.3. Heterologous expression
The coding region of each cDNA was amplified using plasmids
(0.5
template. The 50 uL PCR mixture overlayed with mineral oil also
contained 0.2
M forward primer that contains a 50-CACC up-
stream of the ATG initiation site, 0.2 M reverse primer that spans
lL, 100 ng) prepared from XL1-Blue cells (see Section 5.2) as
l
l
the stop codon, 0.4 mM dNTPs, and 1 unit of Pfu hotstart Turbo
DNA Polymerase (Stratagene). Thermal cycling (95 °C for 3 min,
followed by 30 cycles of 95 °C for 1 min, 55 °C for 1 min and
72 °C for 3 min, ending in a 10 min incubation at 72 °C) was per-
formed on a Robocycler II (Stratagene). PCR products were cloned
into pET101 TOPO cloning vector (Invitrogen) and transformed into
TOP10 cells (Invitrogen) according to the manufacturer’s instruc-
tions. Positive colonies were grown and used as source of plasmids
for heterologous expression. The plasmid inserts were sequenced
to verify correct orientation of the cDNA along the vector’s reading
frame. These plasmids were then transformed into an expression
host BL21DE3 (Invitrogen), following the manufacturer’s protocol.
Overnight cultures of BL21DE3 transformants were inoculated into
5.6. Phylogenetic analysis
Phylogenetic analysis was conducted using the Mega4 software
(Tamura et al., 2007). Deduced amino acid sequences of selected
LOXs were aligned by Thompson et al. (1994) with a gap opening
penalty of 3 and a gap extension penalty of 1.8. Based on the result-
ing multiple alignment, a phylogenetic tree was drawn using the
Neighborhood-Joining method (Felsenstein and Churchill, 1996)
using default parameters in the Mega4 software.
Sequence data
50 mL LB media containing ampicillin (50 lg/mL) and grown until
the absorbance of the culture at 600 nm reached 0.5. Protein
expression was induced by addition of IPTG to a final concentration
of 0.5 mM, and subsequent incubation at 20 °C for 24 h. Cells were
harvested by centrifugation (4000g, 20 min, 4 °C) and pellets were
stored at ꢁ20 °C until use. Protein concentration was determined
using the BCA protein assay kit (Pierce). SDS–PAGE was performed
according to Fuller et al. (2001).
The nucleotide sequences reported in this paper have
been deposited in the GenBank with the following accession
numbers: ABF66647, ABF66648, ABF66649, ABF66650, ABF66651,
ABF66652, and ABF66653.
Acknowledgements
The authors are grateful for the financial support obtained from
The M.J. Murdock Charitable Trust (to H.G.), from the Deutsche
Forschungsgemeinschaft (to I.F.) and from Southern Illinois Uni-
versity SEED Grant (to A.A.).
5.4. Assay of LOX activity
Cell pellets were resuspended in 0.1 M phosphate buffer pH 7.0
(1 mL) and sonicated while on ice (3 ꢂ 15 s, setting 4) using a
Microson ultrasonic cell disruptor (Misonix). After centrifugation
(23,000g, 30 min, 4 °C), the supernatant was collected for subse-
quent LOX assays. Lipoxygenase activity was determined polaro-
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