8/2/2019 león 2002
http://slidepdf.com/reader/full/leon-2002 1/8
Lipoxygenase H1 Gene Silencing Reveals a Specific Role inSupplying Fatty Acid Hydroperoxides for Aliphatic AldehydeProduction*
Received for publication, August 13, 2001, and in revised form, October 22, 2001Published, JBC Papers in Press, October 23, 2001, DOI 10.1074/jbc.M107763200
Jose Leon‡§, Joaquın Royo‡¶, Guy Vancanneyt‡, Carlos Sanz**, Helena Silkowski‡‡,
Gareth Griffiths‡‡, and Jose J. Sanchez-Serrano‡§§
From the ‡Centro Nacional de Biotecnologıa, Consejo Superior de Investigaciones Cientıficas, Campus de Cantoblanco,Universidad Autonoma de Madrid Colmenar Viejo km 15,500, 28049 Madrid, Spain, the ** Instituto de la Grasa,Consejo Superior de Investigaciones Cientıficas, Avenida Padre Garcıa Tejero 4, 41012 Sevilla, Spain,and ‡‡ Horticulture Research International, Wellesbourne, Warwick 35CV 9EF, United Kingdom
Lipoxygenases catalyze the formation of fatty acid hy-droperoxide precursors of an array of compounds involvedin the regulation of plant development and responses tostress. To elucidate the function of the potato 13-lipoxygen-ase H1 (LOX H1), we have generated transgenic potatoplants with reduced expression of the LOX H1 gene as a
consequence of co-suppression-mediated gene silencing.Three independent LOX H1-silenced transgenic lines wereobtained, having less than 1% of the LOX H1 protein pres-ent in wild-type plants. This depletion of LOX H1 has noeffect on the basal or wound-induced levels of jasmonatesderived from 13-hydroperoxylinolenic acid. However, LOXH1 depletion results in a marked reduction in the produc-tion of volatile aliphatic C6 aldehydes. These compoundsare involved in plant defense responses, acting as eithersignaling molecules for wound-induced gene expression oras antimicrobial substances. LOX H1 protein was localizedto the chloroplast and the protein, expressed in Escherichia
coli, showed activity toward unesterified linoleic and lino-lenic acids and plastidic phosphatidylglycerol. The resultsdemonstrate that LOX H1 is a specific isoform involved in
the generation of volatile defense and signaling compoundsthrough the HPL branch of the octadecanoid pathway.
Lipoxygenase (LOX)1 enzymes catalyze the stereospecific di-
oxygenation of unsaturated fatty acids with a 1,4-pentadiene
system. C18 unsaturated fatty acids, linoleic acid (18:29,12)
and linolenic acid (18:39,12,15), are major LOX substrates in
plants. The lipoxygenase pathway of fatty acid metabolism (1)
is initiated by the addition of molecular oxygen at the C9 or C13
position of the acyl chain yielding the corresponding 9- and
13-hydroperoxides (2). Both 9- and 13-hydroperoxides can sub-sequently be cleaved to short-chain oxoacids and aldehydes by
the action of hydroperoxide lyases (HPL) or, alternatively, the
13-hydroperoxide is converted, after enzymatic cyclization, re-
duction, and -oxidation, to JA (3). Plant LOXs are ubiquitous
and encoded by multigene families (4). The presence in a given
tissue of several LOX isoforms with different substrate prefer-
ences, kinetic parameters, stereospecificity in substrate oxy-
genation, pH dependence, and subcellular localization makes
difficult the assignment of specific functions to each LOX iso-
form. Moreover, LOX expression in plants is regulated
throughout development and in response to stress (5, 6). Dif-
ferent LOX isoforms may have different physiological roles;
they may, on the one hand, be responsible for the production of
signals involved in the regulation of plant growth and the
activation of stress-induced defense responses, whereas, on the
other, the products of LOX activity may exert a direct deterring
function toward pests and/or pathogens. It has been proposed
that some aldehydes, in particular hexanal and hexenals pro-
duced by the action of HPL on LOX-derived fatty acid hy-
droperoxides, may be involved in the interaction between
plants and pathogens or parasites (7–10) and, more recently, in
the regulation of wound-induced gene expression (11). JA is
well established as a regulator of defense mechanisms against
wounding, caused by mechanical damage, chewing insects, and
pathogen attack (3, 12–17).
Three LOX gene families have been characterized in potato;
LOX1 is mainly expressed in tubers and roots, whereas LOX2
and LOX3 are expressed in leaves and are wound- and JA-
inducible (18, 19). Several LOX1-derived cDNAs have been
isolated and shown to code for proteins with 9-LOX activity.
LOX2 and LOX3 families are represented each by a single
cDNA, LOX H1 and LOX H3, respectively, encoding proteins
with 13-LOX activity (19). LOX genes with a high degree of
sequence similarity to potato LOX H1 and H3 have been iden-
tified in tomato (20). The expression of the LOX H3 homologue
(TomLoxD) was induced in the leaves of tomato plants by
wounding and methyl jasmonate, following time courses simi-
lar to its potato counterpart. The expression of the tomato LOX
H1 homologue (TomLoxC) was shown to be constitutive in
fruits but, in contrast to potato, it was not detected in either
wounded or nondamaged leaves.
* This work was supported by Spanish Comision Interministerial deCiencia y Tecnologıa Grant BIO99-1225, by Spanish Ministerio deEducacion y Ciencia postdoctoral contracts (to J. L. and J. R.) andpostdoctoral fellowship (to G. V.), and by the Spanish Ministerio deEducacion y Ciencia-British Council Acciones Integradas Program (toJ. J. S.-S. and G. G.). The costs of publication of this article weredefrayed in part by the payment of page charges. This article musttherefore be hereby marked “advertisement” in accordance with 18U.S.C. Section 1734 solely to indicate this fact.
§ Present address: Inst. de Biologia Molecular y Celular de Plantas,Universidad Politecnica de Valencia, Consejo Superior de Investigacio-nes Cientıficas, 46022 Valencia, Spain.¶ Present address: Dept. de Biologıa Celular y Genetica, Universidad
de Alcala de Henares, 28871 Madrid, Spain. Present address: Aventis CropScience, B-9000 Ghent, Belgium.§§ To whom correspondence should be addressed. Tel.: 34-91-
5854500; Fax: 34-91-5854506; E-mail: [email protected] The abbreviations used are: LOX, lipoxygenase; JA, jasmonic acid;
HPL, hydroperoxide lyase; GC, gas chromatography; MGDG, mo-nogalactosyl diacylglycerol; DGDG, digalactosyl diacylglycerol; PE,phosphatidylethanolamine; PC, phosphatidylcholine; PG, phosphati-dylglycerol; TLC, thin layer chromatography; PIN2, proteinase inhibi-tor II; gfw, gram(s) fresh weight; Rubisco, ribulose-bisphosphatecarboxylase/oxygenase; TBS, Tris-buffered saline; Tricine,
N -[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 1, Issue of January 4, pp. 416–423, 2002 © 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
This paper is available on line at http://www.jbc.org416
8/2/2019 león 2002
http://slidepdf.com/reader/full/leon-2002 2/8
Both antisense and co-suppression-mediated depletion of
LOX genes have proved useful tools to elucidate the function of
specific LOX isoforms and their corresponding products in de-
fense signaling. In Arabidopsis, co-suppression-mediated de-
pletion of a specific LOX isoform led to a reduction in the
wound-induced accumulation of JA (21). On the other hand,
antisense-mediated depletion of LOX gene expression has suc-
cessfully been used to establish the involvement of a LOX
isoform in the incompatibility trait of a tobacco variety resist-
ant to the fungus Phytophthora parasitica (22), and the instru-
mental role of LOX H3 in the regulation of wound-induced gene
expression and susceptibility to insect attack in potato (23).
Here we report a transgenic approach to elucidate the func-
tional role of LOX H1 in growth and development of potato
plants, and to assess its potential role in the response to me-
chanical damage. We have generated transgenic lines express-
ing the complete LOX H1 cDNA under the control of the cau-
liflower mosaic virus 35 S promoter. Three transgenic lines
that have undergone silencing of the transgene expression and
co-suppression of the endogenous LOX H1 gene, have been
characterized in terms of plant development and wound-in-
duced changes in gene expression patterns.
EXPERIMENTAL PROCEDURES Plant Material and Transformation —Potato plants ( Solanum tu-
berosum cv. Desiree) were grown in soil either in the greenhouse or in
growth chambers at 22 °C under a 16-h light/8-h darkness photoperiod.Plants were transformed as described (24) by cocultivation with
Agrobacterium tumefaciens harboring, in the BIN19 vector (25), thecomplete LOX H1 cDNA (19) in sense orientation under the control of the 35 S cauliflower mosaic virus promoter and the 3 terminatorsequence of the octopine synthase gene. Plant transformants wereselected for resistance to 50 mg/liter kanamycin (Sigma) and, afterrooting, transferred to soil and grown as described above. Selectedtransformed lines were propagated vegetatively by either tuber sowingor explant cuttings. When indicated, potato leaves were wounded asdescribed previously (19), and plant material harvested at the indicated
times after wounding. RNA and Protein Analysis —Total RNA isolation and Northern blot
techniques were performed as described previously (19). Quantitation
of PIN2 and LOX H3 RNAs was done by densitometry of autoradio-grams derived of four independent experiments, using the Molecular
Analyst program (Bio-Rad). Protein was extracted in 0.1 M Tris-HClbuffer, pH 7.2, containing 20% (v/v) glycerol, and electrophoreticallyseparated in 10% PAGE gels under denaturing conditions (26). Sepa-rated proteins were then transferred to ECL-nitrocellulose membranes(Amersham Biosciences, Inc.), and subsequently hybridized with rabbitantibodies raised against LOX H1 protein or a specific peptide of LOX
H3 as described previously (23), or against proteinase inhibitor 2 (kind-ly donated by Prof. Clarence A. Ryan, Washington State University,Pullman, WA). A peroxidase-coupled goat anti-rabbit antibody wasused to detect immunoreactive proteins by the enhanced chemilumi-nescence system (Amersham Biosciences, Inc.).
Chloroplast Isolation —Twenty grams of potato leaves were har- vested and directly homogenized in a blender in 200 ml of ice-coldextraction buffer (0.35 M sorbitol, 50 mM Tris-HCl, pH 8.0, 5 mM EDTA,0.1% bovine serum albumin, 15 mM -mercaptoethanol). The homoge-nate was filtered through two layers of Miracloth (Calbiochem) andcentrifuged (1000 g) for 10 min at 4 °C. The pellet was gently resus-
pended with a paint brush in 5 ml of ice-cold suspension buffer (0.3 M
sorbitol, 20 mM Tricine-KOH, pH 7.6, 5 mM Mg2
Cl, 2.5 mM EDTA), andpoured onto a Percoll (Amersham Biosciences, Inc.) gradient, preparedon the previous day as follows; 30 ml of 50% Percoll in suspension bufferwere centrifuged 30 min at 43,000 g at 4 °C and kept overnight at4 °C in the centrifuge tubes.
Separation of a layer with intact chloroplasts was achieved after 10min of centrifugation at 13,200 g at 4 °C in a HB-6 rotor (Sorvall). Thechloroplast layer was carefully withdrawn with a glass pipette, and thechloroplasts washed in 20 ml of ice-cold suspension buffer. After 10 minof centrifugation at 2000 g at 4 °C, the chloroplasts in the pellet were
lysed in 1 ml of TE buffer (50 mM Tris-HCl, pH 8.0, 5 mM EDTA), andprotein concentration was determined by the Bradford method(Bio-Rad).
Immunohistochemical Localization of LOX H1 Protein —Wounded
and nonwounded leaflets of potato leaves, from wild-type and LOX H1-co-suppressed transgenic plants, were fixed by vacuum infiltrationof square pieces (5 mm/side) with a 2% (w/v) paraformaldehyde solution
in 0.1 M Tris-HCl buffer, pH 7.2, and then thoroughly washed threetimes with water. After fixation, leaf samples were progressively dehy-drated by incubating for 1 h at room temperature in 30, 50, 70, 85, and100% (v/v) ethanol. Dehydrated tissue was treated twice with xylene for1 h each and then embedded in molten warm paraffin overnight at42 °C. Paraffin blocks, containing tissue samples, were formed by de-
creasing temperature and subsequently used to cut strips of 8-m
sections with a microtome (Leica). Sections were laid on 2% triethox-ysilyl-propylamino-treated glass microscope slides for further process-ing. Before immunolocalization procedure, tissue sections were depar-affinized by immersion twice in xylene and then thoroughly washed
with TBS. Sections were blocked by incubation in TBS containing 1%(w/v) bovine serum albumin and 0.1% (v/v) Tween 20 for 3 h at roomtemperature. After washing three times with TBS containing 0.1% (v/v)bovine serum albumin and 0.1% (v/v) Tween 20 (washing buffer), sec-tions were incubated with a 1:1000 dilution of antibody against LOX H1in washing buffer for 1.5 h. After three new rounds of washing as
described above, tissue sections were incubated in a high humidityclosed plastic chamber with 0.1 ml of a 1:40 dilution of a 5-m goldparticle-coupled anti-rabbit antibody (Sigma) in washing buffer for 1 h.Gold-labeled proteins were then treated with a silver enhancing kit(Amersham Biosciences, Inc.) as described by the manufacturers. La-beled proteins were observed and photographed by using a Nikon mi-
croscope equipped with an IGS filter that allows detection of epipolar-
ized fluorescence. Determination of Endogenous Volatiles and Jasmonates from Potato
Leaves —Twenty-four discs were cut with a 5-mm cork borer from dif-ferent leaves of potato plants, and immediately stored in liquid N
2.
Endogenous volatiles were quantified in quadruplicate by placing 24
leaf discs in an 11-ml vial containing 2.1 ml of saturated CaCl2
solution.The vial was then transferred into an automatic headspace sampler(Hewlett-Packard 19395A), where a 20-min equilibrium time at 80 °Cwas set to allow endogenous volatiles to enter the gas phase. The
volatiles were determined by gas liquid chromatography in a gas chro-
matograph (Hewlett-Packard 5890-II) equipped with a flame ionizationdetector and a glass column (2 mm 1 m) containing 5% Carbowax 20M on 60/80 Carbopack B as the stationary phase. Column temperaturewas held isothermally at 120 °C, injector at 150 °C, and detector at250 °C. Carrier gas (N
2) flow rate was 35 ml/min. Quantitation was
performed by conversion of peak areas into nanomoles produced by the
24 leaf discs in 30 min by means of calibration curves obtained for the
different compounds.Jasmonate contents in nonwounded and damaged leaves were deter-
mined by competitive enzyme-linked immunosorbent assay as de-scribed (23).
LOX H1 Production in E. coli —LOX H1 synthesis in BL21 E. colicells and assays for LOX activity were essentially performed as de-scribed (19) with the exception that 20% glycerol and 0.01 mM phenyl-methylsulfonyl fluoride were included in the buffer. LOX activity was
determined spectrophotometrically by monitoring the increase in A234
resulting from conjugated diene formation from exogenously suppliedlipid substrates containing polyunsaturated fatty acids provided asunesterified compounds or esterified to complex lipids extracted frompotato leaf as described below. Typical assays contained 2–10 l of bacterial lysate supernatant, fatty acid, or complex lipid substrate(10 –50 nmol, in ethanol, 0.01% final concentration) in 100 mM sodiumacetate buffer, pH 6.0, at 25 °C.
Lipid Analysis and Substrate Preparation —Extractions were per-formed using chilled solvents and glassware at 4 °C. Lipids were ex-tracted from 2 g of fresh leaf material in chloroform/methanol/0.15 M
acetic acid (10/20/7.7, v/v) in a pestle and mortar and then an additional10 ml of chloroform and 10 ml of water were added to achieve phaseseparation. The lower chloroform phase containing the complex lipidswas removed and reduced to dryness under nitrogen. The residue wasresuspended in a small volume of chloroform and the complex lipidsseparated by TLC in a chloroform/methanol/acetic acid/water (170/30/ 20/7, v/v). For quantitation, the complex lipids were transmethylated insitu in the silica gel using 2.5% sulfuric acid in methanol and theresulting methyl esters extracted into hexane and analyzed by GCusing heptadecanoic acid as internal standard (27). For the preparationof lipid substrates for studies on LOX H1, after TLC, the lipids were
eluted from the silica gel using the following solvents: acetone (100%)for DGDG, chloroform/acetone (50/50, v/v) for MGDG, chloroform/meth-anol (80/20, v/v) for PE, and chloroform/methanol (50/50, v/v) for PG and
PC (28). For larger scale production of PG, complex lipids were ex-
LOX H1-depleted Transgenic Potato Plants 417
8/2/2019 león 2002
http://slidepdf.com/reader/full/leon-2002 3/8
tracted from 10 g of tissue and fractionated using DEAE column chro-matography (28). The authenticity of PG was verified by TLC purifica-tion and GC analysis confirming the presence of its characteristic fattyacid, 16:1 (trans-3). The lipids were dried under nitrogen and resus-
pended in ethanol and stored under an atmosphere of nitrogen at20 °C until required.
RESULTS
Silencing of LOX H1 Gene in Transgenic Potato Plants —
Previously we have cloned and characterized a systemically
induced 13-lipoxygenase (LOX H1) from potato leaves that is
transcriptionally up-regulated in both wounded and nondam-
aged tissues (19). To ascertain the functional role of LOX H1,
overexpression of the LOX H1 gene in transgenic potato plants
was undertaken using a full cDNA under the control of the
cauliflower mosaic virus 35 S promoter. Forty-eight independ-
ent transgenic lines were generated, and their basal level of
LOX H1 transcript was compared with that of wild-type plants.
Three of these lines (lines 9, 31, and 33) had much reduced LOX
H1 transcript levels in nonwounded leaves (Fig. 1 A), which was
suggestive of silencing or co-suppression of both transgene and
endogenous gene expression. We tested whether silencing of
LOX H1 gene in co-suppressed plants also affected its wound-induced expression. Local and systemic induction of LOX H1
expression upon wounding was detected in wild-type plants but
was almost completely suppressed in the three transgenic lines
analyzed (Fig. 1 B).
We also tested whether LOX H1 silencing affects the expres-
sion of LOX H3, another wound-inducible 13-lipoxygenase from
potato leaves (19, 23). Fig. 2 A shows that LOX H3 expression is
induced upon wounding in the LOX H1-co-suppressed lines.
Although in these plants LOX H3 transcript levels were lower
than in the wild-type ones, the level of induction of LOX H3
gene expression in wild-type and co-suppressed plants was
similar because of its lower basal transcription in the latter
(Fig. 2 B). However, the wound-induced accumulation of pro-
teinase inhibitor 2 ( PIN2) transcript was reduced in all LOX
FIG. 2. Expression of wound-induc-ible genes in wild-type and LOX H1-co-suppressed potato plants. A, totalRNA was isolated from wounded leafletsof wild-type (WT ) and LOX H1-co-sup-pressed plants (#9, #31, and #33) at theindicated times (in hours) after wounding,and the levels of the transcripts from dif-ferent wound-inducible genes were ana-lyzed by Northern blot techniques using32P-labeled probes corresponding to li-poxygenases ( LOX H1 and LOX H3), andproteinase inhibitor II ( PIN2) cDNAs.Equal RNA loading was verified byethidium bromide staining of the riboso-mal RNA (rRNA) in the gel. B, quantita-tion of LOX H3 and PIN2 transcript accu-
mulation in leaves of wild type ( emptysquares or circles) and LOX H1-co-sup-pressed line 31 ( full squares or circles)upon wounding. RNA samples were takenat different times (in hours) after wound-ing. Values represent -fold induction overthe levels determined in the nonwoundedleaves of the corresponding plants and arethe mean (bars represent standard devi-ations) of values obtained in four inde-pendent Northern blot assays.
FIG. 1. Analysis of LOX H1-silenced transgenic potato plants. A, Northern analysis of independent transgenic lines. Total RNAs wereisolated from nonwounded leaves of transgenic plants and a non transformed plant (wt) as a control, and hybridization to the 32P-labeled NotIfragment containing the complete LOX H1 coding sequence was performed to detect endogenous gene and transgene transcripts, having the samesize. B, wound-induced expression of LOX H1 gene. LOX H1 transcript was analyzed at different times in hours after wounding (h.a.w.) byNorthern techniques with total RNA isolated from either wounded leaflets (local) or the nonwounded leaflets (systemic) of wounded leaves of wild-type (wt) and LOX H1-co-suppressed lines (#9, #31, and #33). Ethidium bromide-stained ribosomal RNA (rRNA) is shown as loading control.
LOX H1-depleted Transgenic Potato Plants418
8/2/2019 león 2002
http://slidepdf.com/reader/full/leon-2002 4/8
H1-co-suppressed lines (Fig. 2 A). A more detailed analysis of
line 31, the one showing the lowest LOX H1 levels (see below),
revealed that wound-induced PIN2 transcript accumulation is
similar up to 6 h but significantly lower at long times after
wounding (24 and 48 h; Fig. 2 B). These data indicate that LOX
H1 is involved in signaling the wound-dependent PIN2
activation.
The severely reduced accumulation of LOX H1 transcript in
the co-suppressed plants prompted us to evaluate to what
extent gene silencing had also led to a reduced accumulation of
the corresponding protein product. Fig. 3 A shows that, despite
the increase in LOX H1 transcript accumulation observed upon
mechanical damage, LOX H1 protein levels were not up-regu-
lated in wounded leaves and remained nearly constant within
the time frame analyzed, probably reflecting a balance between
increase in transcription rate and protein turnover. Alterna-
tively, translation of LOX H1 transcripts accumulating upon
wounding may be delayed or inefficient within the time frame
analyzed. Transgenic lines 9, 31, and 33, which did not accu-
mulate LOX H1 transcript upon wounding (Figs. 1 B and 2),
had undetectable levels of protein whereas strong expression
was seen in extracts from wild type plants (Fig. 3 A). Only after
long exposure times were residual levels of a cross-reacting
protein detected in extracts from wounded leaves of co-sup-pressed plants. Quantitation of these residual levels indicated
that line 33 had some 50 times less LOX H1 protein than
wild-type plants by 24 h after wounding (Fig. 3 B). Silencing of
LOX H1 gene expression was more efficient in lines 9 and 31.
The levels of LOX H1 protein in the wounded leaves of these
lines were at least 100 times lower than in wild-type plants
(Fig. 3 B). In agreement with the levels determined for their
corresponding transcripts, the reduced levels of LOX H1 pro-
tein in co-suppressed plants did not affect the levels of LOX H3
protein and resulted in a slight reduction of PIN2 protein levels
(Fig. 3 A).
Developmental Control of LOX H1 Gene Silencing in Potato
Plants — LOX H1 gene silencing in leaves of the co-suppressed
transgenic line 31 appears to be subject to developmental reg-
ulation, as indicated by the analysis of LOX H1 protein con-
tents in the foliage, from actively growing apical leaves to adult
fully expanded ones. In contrast to wild-type plants in which,
as shown in Fig. 4, LOX H1 is constitutively present at similar
levels in leaves at different developmental stages, LOX H1
gene expression appears to be silenced in fully expanded leaves
(Fig. 4, lanes 3 –7 , #31 leaf ) but only partially suppressed in
actively growing, developing leaves (Fig. 4, lanes 2 and 3, #31
leaf ) of line 31.
LOX H1 gene is also expressed in flowers from potato plants
(19) and the levels of protein do not change significantly at
different stages of flower development (Fig. 4). We observed
that gene silencing also occurred in the flowers of co-sup-
pressed plants and was maintained throughout flower devel-
opment (only shown for line 31).
Phenotypic Characterization of LOX H1-co-suppressed Potato
Plants —The pattern and levels of LOX H1 expression sug-
gested that it may have a function in plant development. In-
deed, lines 9 and 31 have shorter internodes than wild-typeplants, and smaller leaves, and branched and bushy plant
shoots in which the axillary buds sprouted often (data not
shown). As this altered phenotype is present in two independ-
ent co-suppressed lines, it likely relates to LOX H1 depletion
and not to the result of somatic variations arising during the
transformation procedure. However, line 33 did not exhibit any
of these differences, perhaps because of its higher content in
LOX H1 relative to lines 9 and 31. No alteration was observed
in the root system of LOX H1-co-suppressed plants (data not
shown). We also analyzed the production of tubers in wild-type
and co-suppressed plants. Plants either grown from tubers or
from explants were able to tuberize, and the total yield and
number of tubers per plant in wild-type and co-suppressed
plants were not significantly different (data not shown).
FIG. 3. Western analysis of lipoxygenase (LOX H1 and LOX H3)and proteinase inhibitor 2 (PIN2) proteins in wounded potatoleaves from wild-type and LOX H1-co-suppressed plants. A, pro-teins were isolated from leaves of wild-type (wt) and LOX H1-co-sup-pressed lines (#9, #31, and #33) at different times after wounding (inhours, h.a.w.), separated in 10% SDS-PAGE, transferred to nitrocellu-lose membranes, and probed with antibodies that cross-react specifi-
cally to LOX H1, LOX H3, and PIN2. B, dilutions, from 1/50 to 1/1000,of the protein preparation corresponding to wt 24 h of panel A were usedto compare the levels of the LOX H1 protein with those present in theleaves of silenced transgenic plants 24 h after wounding. Detection of the residual protein was possible by using at least 15 times longerexposure than that used for Westerns shown in panel A.
FIG. 4. Developmental control of LOX H1 depletion in LOX H1-co-suppressed potato plants. Proteins were extracted from greenflower buds (3 mm in diameter, lane 1) and from different leaves of increasing age, from the apex (lane 2) to fully expanded adult leaves(lane 8) of wild-type (WT ) plants and the LOX H1-co-suppressed line 31.Proteins were also extracted from wild-type and line 31 flowers. Lane 1,green buds; lane 2, green buds (6 mm in diameter) with emergingpetals; lane 3, buds (8 mm in diameter) with colored petals; lane 4,adult, open flower; lane 5, senescing flower. Western-type assay usingspecific antibodies to LOX H1 was conducted to determine LOX H1protein content. The autoradiogram shown for line 31 flowers required8 –10-fold longer exposure times.
LOX H1-depleted Transgenic Potato Plants 419
8/2/2019 león 2002
http://slidepdf.com/reader/full/leon-2002 5/8
Immunolocalization of LOX H1 in Potato Leaves — Analysisof the deduced amino acid sequence of LOX H1 protein
prompted the suggestion that LOX H1 gene product should be
localized inside chloroplasts (19). We have now addressed the
localization of LOX H1 by immunohistological techniques. The
results shown in Fig. 5 indicate that, indeed, LOX H1 protein
specifically accumulates in chloroplasts both in nonwounded
and in damaged potato leaves (Fig. 5, C – F ) and also that no
LOX H1 protein was detected in leaves of the co-suppressed
line 31 (Fig. 5, G and H ). The specific detection of LOX H1
protein was confirmed using the pre-immune serum that gave
no signal in the immunolocalization procedure (Fig. 5, A and
B). No LOX H1 protein outside the chloroplasts was detected in
palisade parenchyma cells nor was it detected in epidermal
cells (shown in the 100-fold magnification of Fig. 5, I and J ).
Consistent with the data obtained in Western-type experi-
ments (Fig. 3), no major wound-induced changes in LOX
H1 protein amount were observed (compare panels D and F in
Fig. 5).
Chloroplastic localization of LOX H1 was confirmed by iso-
lating intact chloroplasts from potato leaves and Western blot
techniques (Fig. 6). As expected, chloroplasts isolated from the
LOX H1-co-suppresed line 31 had a much reduced content of
LOX H1 protein.
Substrate Specificity of Recombinant LOX H1 Protein —It is
generally assumed that the substrates for LOX are unesterified
polyunsaturated fatty acids released from complex lipids by the
action of lipases and recently a role for phospholipase A2 acting
on PC, analogous to mammals, has been proposed in plants (29,
30). However, LOX activity toward complex lipids has also been
reported (4). In potato leaves the major complex lipids present
are the galactolipids, MGDG (58.6 1.5%) and DGDG (24.8
1.4%), with the three main phospholipids being PC (7.6
1.1%), PE (4.4 0.6%), and PG (4.4 0.9%). PC and PE are
predominantly found in the endoplasmic reticulum, whereas
PG is the only phospholipid exclusively synthesized and located
within the chloroplast (31). All complex lipids contained signif-
icant levels of 18:3, the fatty acid precursor of JA although 16:3,
the precursor of dinor-JA (32) was a major acyl constituent in
only MGDG and PG (Table I). These major complex lipids were
purified from potato leaves in sufficient quantity necessary forsubstrate specificity studies of the cloned LOX H1.
LOX H1 activity in extracts from transformed bacterial
strains was highest toward the unesterified fatty acid sub-
strates, linoleic acid and linolenic acid, which were utilized at
similar rates (46.7 and 52.9 nmol of hydroperoxylinolenic acid
formed/min/mg, respectively). When complex lipids were of-
fered as substrates, no activity was detected with MGDG,
DGDG, or PE. However, significant rates of activity were ob-
served with PG, and in three independent preparations the
rate was 16.7 9.5% of that observed for 18:3. Some activity
toward PC was also observed but was 10 –20% of that seen with
PG as substrate. The activity of a commercially available li-
poxygenase from soybean (Sigma) on PG as a substrate was
also examined. Although good rates of activity were evident
FIG. 5. Immunolocalization of LOX H1 protein in potatoleaves. Leaf sections were incubated with a 1:1000 dilution of antibodyagainst LOX H1 (C – J ) or a pre-immune serum ( A and B) as primaryantibody, and a 1:40 dilution of a 5-m gold particle-coupled anti-rabbitantibody as secondary antibody. Green spots correspond to brightnessoriginated from silver-enhanced gold-labeled proteins visualized withan IGS filter that allows detection of epipolarized fluorescence. A – D,nonwounded wild-type leaf sections. E, F , I , and J , wounded leaves (24h after wounding) of wild-type plants. G and H , wounded leaves (24 hafter wounding) of LOX H1-co-suppressed plants (line 31). Immuno-staining was detected by illumination with epipolarized light ( B, D, F ,
H , and J ) or a combination of epipolarized light with standard whitelight ( A, C, E, G, and I ). Tissue was stained with aniline blue. Originalmagnifications, 40 ( A – H ) and 100 ( I and J ).
FIG. 6. LOX H1 content in chloroplasts. LOX H1 content in pro-tein extracts from total leaf tissue (T ) and isolated chloroplasts (C) fromwild-type (WT ) and LOX H1-co-suppressed plants (#31) was comparedby Western blotting using specific antibodies to LOX H1 (right panel).
A replica gel was stained with Coomassie Blue (left panel) to showprotein content and complexity of the respective extracts. The most
intensely stained band corresponds to the Rubisco large subunit, whichlocalizes to the chloroplast stroma. Molecular sizes (in kilodaltons) aremarked on the left. The position of LOX H1 protein is marked on theright.
LOX H1-depleted Transgenic Potato Plants420
8/2/2019 león 2002
http://slidepdf.com/reader/full/leon-2002 6/8
with unesterified fatty acids, no activity toward any complex
lipid, including PG, could be demonstrated (data not shown).
The Role of Fatty Acid Hydroperoxides Produced by LOX
H1 —Lipoxygenases produce fatty acid hydroperoxides precur-
sors for an array of different compounds (33). Biosynthesis of
JA requires a 13-lipoxygenase activity (3). As LOX H1 cata-
lyzes this type of reaction (19), we assessed the effect of LOX
H1 depletion on basal and wound-induced JA levels in potato
leaves. Jasmonate contents in the leaves of LOX H1-co-sup-
pressed potato plants, as determined by competitive immuno-assay (23), were not significantly different from those in wild-
type plants both prior to and after wounding. A jasmonate
content of 1.23 0.9 nmol/gfw was determined in nondamaged
leaves of wild-type plants, and this level rose to 3.05 0.8
nmol/gfw 6 h after wounding. In the LOX H1-co-suppressed
lines 9 and 31, the corresponding values in nondamaged leaves
were 0.75 0.4 and 0.48 0.1 nmol/gfw, respectively, and
2.96 0.2 and 5.29 2.1 nmol/gfw 6 h after wounding. Al-
though these results do not exclude its participation in jas-
monate metabolism, they suggest that LOX H1 is not involved
in the bulk production of jasmonates upon wounding.
In addition to serving as precursors for jasmonate synthesis,
13-hydroperoxides of both linoleic and linolenic acids can be
cleaved by HPL, yielding the C-6 aldehydes, hexanal and Z,3-hexenal. We have determined the levels of these aldehydes, and
the alcohols derived from them, in the leaves of wild-type and
LOX H1-co-suppressed plants (lines 31 and 33). The results,
summarized in Table II, show that plants with silenced expres-
sion of LOX H1 have less than 7 and 2% of hexanal and
E,2-hexenal (the more stable isomer of Z,3-hexenal), respec-
tively, of the levels determined for wild-type plants. Z,3-hex-
enol, which is the product of further reduction of E,2-hexenal
by alcohol dehydrogenase, is present in lines 31 and 33 at less
than 5% of the wild-type level, confirming that LOX H1-si-
lenced plants are compromised in the production of C6-alde-
hydes and alcohols from the heterolytic cleavage of 13-hy-
droperoxides of linoleic and linolenic acids. In contrast, hexanal
and E,2-hexenal levels in plants depleted for a different wound-
responsive 13-LOX ( LOX H3; line H3– 4; Ref. 23) were similarto those found in wild-type plants. We have also determined the
levels of C5-aldehydes and -alcohols, 1-penten-3-ol and Z,2-
pentenol, which are further oxidized to ethyl vinyl ketone and
Z,2-hexenal, derived from lipoxygenase-mediated cleavage of
13-hydroperoxides of linoleic and linolenic acids (34). Those
intermediates are present in H3– 4 plants at around 80% of the
levels detected in wild type plants, but all of them were signif-
icantly reduced in LOX H1-silenced plants. These data indicate
that, in contrast to LOX H3, LOX H1 is an essential enzyme for
the generation of C6- and C5-aldehydes and alcohols.
DISCUSSION
Fatty acid hydroperoxides and their metabolic derivatives
(35, 36) are proposed to participate in the plant defense re-
sponse either as signaling compounds for activation of defense
genes or as deterrents of insect attack and pathogen prolifera-
tion. In potato leaves, 13-hydroperoxides are generated by at
least two different 13-LOX activities encoded in gene families
( LOX2 and LOX3) with distinct patterns of induced expression
upon mechanical damage. Antisense-mediated suppression of
one of them ( LOX3) showed that a specific LOX isoform (LOX
H3) is required for mounting an efficient defense against insect
herbivores, through induction of proteinase inhibitors and
other defense-related genes (23). In this work, co-suppressionof LOX2 gene expression, encoding a second 13-LOX isoform
(LOX H1), reveals that the most likely role of LOX H1 is to
supply HPL with the 13-hydroperoxy fatty acid substrates for
the production of C-6 aldehydes, such as hexanal and 3-hex-
enal, and C-12 oxoacids. It has recently been shown that aphids
grown in transgenic potato plants in which HPL has been
depleted exhibit a much better performance than those main-
tained in wild-type plants (37). Because both LOX H1 and HPL
are present in the leaves of healthy plants; this pathway is thus
a constitutively deployed defense response toward sucking
insects.
Suppression of gene expression in transgenic plants stands
as the method of choice for ascribing specific functions to gene
products displaying similar activities. LOX H1-co-suppressedplants have provided a good model for the elucidation of its
functional role because of the high selectivity of the co-suppres-
sion effect. Indeed, although LOX H1-co-suppressed plants
have less than 1% of the protein detected in wild-type plants,
they have standard levels of LOX H3 that is also induced in
leaves in response to wounding (19).
The LOX H1-depleted potato plants were obtained in an
experiment designed to overexpress LOX H1 driven by the
strong 35 S promoter from the cauliflower mosaic virus. Sur-
prisingly, in none of the transformed lines could any signifi-
cantly higher level of LOX H1 transcript accumulation be de-
tected. In fact, three of them showed a drastic decrease in the
level of LOX H1 transcript and protein, suggesting that trans-
gene-mediated co-suppression of LOX H1 gene expression oc-curred. These observations suggest that potato plants may not
tolerate high LOX H1 levels, which could compromise the via-
bility of the plant by so far unknown mechanisms. As silencing
of LOX H1 gene expression is observed in three independent
transgenic lines, it can confidently be ascribed to the action of
the introduced transgene.
Co-suppression of LOX H1 leads to depletion of the protein in
leaves and flowers of potato plants, but, remarkably, silenced
expression was less evident in young actively growing apical
leaves. Developmental regulation of silencing has already been
reported (38). Although we cannot rule out the possibility that
the developmental effects on LOX H1 co-suppression may ac-
tually be mediated by changes in the 35 S promoter activity (39,
40), limited co-suppression in actively growing leaves may in-
T ABLE I Fatty acid composition of the major complex lipids in wild type potato leaves
Lipids were extracted from freshly harvested leaf material in an acidified chloroform/methanol-based solvent (27). Lipids were purified by TLCand quantified as their fatty acid methyl esters by GC using heptadecanoic acid as internal standard. Values are presented as the mean of threeindependent samples standard error. Tr, trace amounts (0.5%). ND, not detected.
LipidFatty acid composition (mol %)
16:0 16:1 16:2 16:3 18:0 18:1 18:2 18:3
mol %
MGDG 1.9 0.4 Tr Tr 31.7 2.7 Tr Tr 2.4 0.6 63.3 2.9
DGDG 7.8 0.9 Tr Tr 2.9 0.5 1.5 0.5 0.5 0.2 2.0 0.9 84.8 2.5PG 15.1 1.5 19.5 2.6 Tr 27.1 3.1 Tr 1.1 0.4 13.6 1.5 23.3 1.7PC 21.2 2.0 ND ND ND 2.3 0.9 5.2 1.4 45.0 3.0 26.0 2.8PE 22.8 1.2 ND ND ND 0.9 0.3 0.7 0.3 51.7 2.3 23.4 2.7
LOX H1-depleted Transgenic Potato Plants 421
8/2/2019 león 2002
http://slidepdf.com/reader/full/leon-2002 7/8
deed result from an essential role of LOX H1 at this develop-
mental stage that requires (and tolerates) higher levels of the
protein.
In support of a function for LOX H1 in regulating growth and
development, we have found that the aerial parts of some of the
LOX H1-co-suppressed lines display a characteristic phenotype
clearly distinguishable from that of wild-type plants. LOX H1-
co-suppressed shoots are smaller and more branchy than wild-
type ones. A high number of actively growing axillary buds
present in the co-suppressed shoots suggests that apical dom-inance is reduced in LOX H1-co-suppressed lines. This pheno-
typic alteration may be because of a direct effect of depleting
any LOX H1-derived products or, alternatively, to a secondary
effect mediated by alterations in the hormonal imbalance in the
co-suppressed plants, as this phenotype resembles that of mu-
tants or transgenic plants affected also in the balance of cyto-
kinins, auxins, and/or brassinosteroids (Refs. 41– 43; for a re-
cent review on apical dominance and control of axillary bud
growth, see Ref. 44). However, because one of the LOX H1-
depleted lines (line 33) does not show these phenotypic traits, it
is unclear to what extent they may be directly related to the
lower LOX H1 levels present in the other transgenic lines (lines
9 and 31).
LOX H1 constitutively accumulates throughout flower devel-
opment, and a role in supplying fatty acid hydroperoxide pre-
cursors for the production of volatiles to attract pollinators
could be envisaged. LOX H1-co-suppressed plants tend to
flower earlier than wild-type ones (data not shown). Remark-
ably, the early flowering Arabidopsis mutant efs has also a
reduction in apical dominance (45), supporting a possible link
between these processes and suggesting that a LOX H1-derived
product may participate in their regulation.
The presence of putative transit peptides in their deduced
protein sequences suggested that LOX H1 resides in plastids
(19). Indeed, chloroplast protein extracts are enriched in LOX
H1 and, moreover, immunolocalization confirms the presence
of LOX H1 exclusively in the chloroplasts. Immunohistology
with specific antibodies also reveals that LOX H3 preferen-
tially accumulates in chloroplasts (data not shown). In thisregard, both potato 13-LOX have a similar subcellular localiza-
tion to their corresponding homologues in tomato (20). Chloro-
plast localization is an important feature regarding the possi-
ble role of 13-LOX in the octadecanoid pathway (1). It has been
proposed that chloroplasts are the major site for fatty acid
hydroperoxide metabolism (46). Other wound and/or jas-
monate-inducible lipoxygenases such as those of barley leaves
are, as well, localized in the chloroplasts (47) or, in the case of
the Arabidopsis AtLOX2, have typical chloroplast transit pep-
tides (6). Because of its 13-LOX stereospecific activity using
linoleic and linolenic acids as substrates, in addition to the
transcriptional activation detected both in wounded and JA-
treated potato leaves (19), both LOX H1 and H3 were good
candidates to be involved in jasmonate synthesis in vivo. We
reported previously (23) that, despite its prominent role in
plant resistance to pest attack, LOX H3 was not a rate-limiting
activity in the wound-induced synthesis of JA. Our results
indicate that wound-induced accumulation of jasmonates is not
reduced in LOX H1-co-suppressed plants either, suggesting
that LOX H1 is not implicated in the synthesis of the bulk of
jasmonates in response to wounding. Instead of serving as JA
precursors, LOX H1-derived fatty acid hydroperoxides are sub-
strates for HPL and, thus, LOX H1-co-suppressed plants have
much reduced levels of the oxylipins produced through thisbranch of the octadecanoid pathway, namely C6 aldehydes and
alcohols and the corresponding oxoacid, traumatic acid. In con-
trast, C-6 aldehyde levels in LOX H3-depleted plants are
nearly identical to wild-type. As both LOX H1 and H3 localize
to chloroplasts, the hydroperoxide pool generated by LOX H1
should be compartmentalized for its exclusive use through the
HPL catabolic pathway, whereas JA synthesis would depend
on the hydroperoxide products of LOX H3 or another, hitherto
uncharacterized, LOX activity. This compartmentalization
could either depend on specific protein-protein interactions to
generate metabolic chains or be because of a differential sub-
organellar distribution of the enzymes involved, thus restrict-
ing access to one another’s hydroperoxide pools. Both possibil-
ities are currently being explored.
It is generally assumed that the substrate for LOX is unes-
terified polyunsaturated fatty acids released from complex lip-
ids by the action of lipases. However, LOX activity toward
complex lipids has also been reported (4). Although unesteri-
fied fatty acids are the preferred substrate for the LOX H1
enzyme in vitro, it also shows significant activity on PG, the
only phospholipid entirely synthesized within plastids (31).
Because LOX H1 is targeted to the chloroplast, we considered
that the galactolipids, which are the major thylakoid complex
lipids and which are rich in 18:3, could be a suitable substrate.
However, no activity toward these lipids was detected, suggest-
ing that this LOX isoform does not act on the major membrane
components of the chloroplast. LOX H1 activity on PG would
thus generate hydroperoxides that are esterified to complex
lipids. To be further processed down the octadecanoid pathway,such ester-linked hydroperoxides would have to be released by
the action of a lipase with a specificity toward oxygenated fatty
acids. In plants such an activity has been observed in the
remodeling of PC by phospholipase A2 in tissues that accumu-
late high levels of the hydroxy-fatty acid, ricinoleic acid, in
their seed oils (48). The significance of this activity of LOX H1
remains, at present, unclear, although the ability of a LOX to
utilize this substrate offers a potential means of compartmen-
tation and restriction of substrate to a relatively minor plastid
phospholipid. However, whether the enzyme uses unesterified
polyunsaturated fatty acids or polyunsaturated fatty acids es-
terified in PG will be dependent on the availability of both
substrates to the enzyme in vivo. Conconi et al. (29) estimated
that unesterified fatty acids levels increased from
75 to
125
T ABLE IIContent of C6- and C5-aldehydes and alcohols derived from cleavage of 13-hydroperoxides of linoleic and linolenic acids in wild type and
LOX-silenced transgenic plants
Nonwounded leaves of wild-type potatoes, and transgenic, LOX H3-silenced (H3 – 4; Ref. 23) and LOX H1 co-suppressed (lines 31 and 33) plantswere used. Values presented are expressed in nmol/cm2 of leaf area, and represent the mean of five independent samples containing 24 leaf discs(5 mm in diameter) each standard error.
Wild-type H3–4 Line 31 Line 33
Z,3-Hexenol 0.53 0.08 0.32 0.07 0.02 0.01 0.01 0.00 E,2-Hexenol 0.046 0.023 0.020 0.005 0.011 0.005 0.007 0.002
Hexanal 0.48 0.11 0.35 0.09 0.03 0.01 0.03 0.01 E,2-Hexenal 13.59 2.40 11.64 1.39 0.16 0.08 0.20 0.10 Z,2-Pentenol 0.41 0.07 0.33 0.05 0.06 0.01 0.05 0.01 Z,2-Pentenal 1-penten-3-ol 0.28 0.06 0.22 0.03 0.06 0.01 0.04 0.01Ethyl vinyl ketone 0.31 0.03 0.26 0.04 0.07 0.03 0.15 0.03
LOX H1-depleted Transgenic Potato Plants422
8/2/2019 león 2002
http://slidepdf.com/reader/full/leon-2002 8/8
g/g dry weight 1 h after wounding and accounted for less than
0.25% of total fatty acids present in tomato leaves. Concomi-
tantly, an increase in lyso-PC was observed suggesting that the
18:3 liberated for subsequent JA synthesis may arise from PC.
In LOX H1-co-suppressed plants, however, no effect of trans-
gene expression on basal or wound-induced levels of JA was
observed. However, the severe reduction in volatile production
observed in the LOX H1-co-suppressed plants suggests that the
pool of lipid hydroperoxides generated by LOX H1 serve as
substrates for HPL. Recently, we have shown that the basal
level of hydroperoxides in potato leaf was 334 75 nmol gfw
and are esterified to complex lipids (27). Thus, the substrate
specificities of LOX H1 reported here suggest that PG could
provide the hydroperoxide fatty acid substrate for HPL lyase
leading to aliphatic aldehyde production.
Both Western blotting and immunohistological detection in-
dicate that LOX H1 accumulates at fairly high levels in non-
wounded potato leaves. However, 9-LOX appears to be the
predominant specific activity in the leaves of healthy, nondam-
aged potato plants (49). LOX H1 may thus be present in an
inactive form in those leaves, and be post-translationally acti-
vated upon stress, or in other situations. LOX H1 could thus be
involved in a defense response to pests and pathogens, different
from that related to herbivory and involving activation of
genes, proteinase inhibitors in particular. Consistent with that
hypothesis, the wound-induced activation of LOX H3 gene and
other wound-responsive genes such as allene oxide synthase
( AOS; data not shown) is not significantly affected in LOX
H1-co-suppressed plants. However, wound induction of the pro-
teinase inhibitor II ( Pin2) gene is partly reduced, perhaps
reflecting the requirement for its full activation of a component
that is not present in LOX H1-co-suppressed plants. It has been
reported that C6 aldehydes may induce a subset of defense-
related genes such as those encoding enzymes of the phenyl-
propanoid pathway (11). However, reducing C6 aldehyde con-
tent through HPL depletion does not result in reduced levels of
Pin2 transcripts upon wounding (37). Thus, LOX H1-derived
compounds other than C6 aldehydes, or LOX H1 itself, may
additionally play a role in the wound-induced activation of a
subset of responsive genes. The use of NADPH oxidase inhib-
itors has revealed that hydrogen peroxide is required for the
induction of a subset of wound-responsive genes, Pin2 among
them, but not for wound-induced activation of LOX H3 and
AOS gene expression (50). Similar to NADPH oxidase inhibi-
tion, the wound-induced activation of both subsets of genes is
affected differentially in the LOX H1-co-suppressed plants,
suggesting that LOX H1 activity is involved in gene induction
through the H2O2-dependent pathway.
The use of LOX H1-co-suppressed potato plants provides
further insights on the role of LOX H1 in the synthesis of
volatiles that are detrimental to pests and pathogens, as a
prerequisite for genetic manipulation of plants toward highlevels of active LOX H1.
Acknowledgments —We gratefully acknowledge Tomas Cascon andPilar Paredes for excellent technical assistance, and Ines Poveda and
Angel Sanz for the photographic work. We thank Prof. C. A. Ryan forpotato pin2 antibodies, M. Dolores Gomez and Luis Canas (IBMCP,
Valencia, Spain) for the assistance in immunohistology techniques, andCarmen Castresana for comments and suggestions on the manuscript.
REFERENCES
1. Vick, B. A. (1993) Lipid Metabolism in Plants (Moore, T. S., ed) pp. 167–191,CRC Press Inc., Boca Raton, FL
2. Rosahl, S. (1996) Z. Naturfosch. C51, 123–1383. Leon, J., and Sanchez-Serrano, J. J. (1999) Plant Physiol. Biochem. 37,
373–3804. Siedow, J. N. (1991) Annu. Rev. Plant Physiol. Plant Mol. Biol. 42, 145–1885. Bell, E., and Mullet, J. E. (1991) Mol. Gen. Genet. 230, 456 –4626. Bell, E., and Mullet, J. E. (1993) Plant Physiol. 103, 1133–11377. Croft, K. P. C., Juttner, F., and Slusarenko, A. J. (1993) Plant Physiol. 101,
13–248. Deng, W., Hamilton-Kemp, T. R., Nelson, M. T., Andersen, R. A., Collins, G. B.,
and Hildebrand, G. F. (1993) J. Agric. Food Chem. 41, 506 –5109. Hildebrand, D. F., Brown, G. C., Jackson, D. M., and Hamilton-Kemp, D. R.
(1993) J. Chem. Ecol. 19, 1875–188710. Vaughn, S. F., and Gardner, H. W. (1993) J. Chem. Ecol. 19, 2337–234511. Bate, N. J., and Rothstein, S. J. (1998) Plant J. 16, 561–56912. Farmer, E. E., Johnson, R. R., and Ryan, C. A. (1992) Plant Physiol. 98,
995–100213. Blechert, S., Brodschelm, W., Holder, S., Kammerer, L., Kutchan, T. M., Xia,
Z.-Q., and Zenk, M. H. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 4099– 410514. Creelman, R. A., and Mullet, J. E. (1997) Annu. Rev. Plant Physiol. Plant Mol.
Biol. 48, 355–38115. McConn, M., Creelman, R. A., Bell, E., Mullet, J. E., and Browse, J. (1997)
Proc. Natl. Acad. Sci. U. S. A. 94, 5473–547716. Vijayan, P., Shockey, J., Levesque, C. A., Coook, R. J., and Browse, J. (1998)
Proc. Natl. Acad. Sci. U. S. A. 95, 7209 –721417. Schweizer, P. A. B., Dudler, R., and Metraux, J.-P. (1998) Plant J. 14, 475–48118. Geerts, A., Feltkamp, D., and Rosahl, S. (1994) Plant Physiol. 105, 269 –27719. Royo, J., Vancanneyt, G., Perez, A. G., Sanz, C., Stormann, K., Rosahl, S., and
Sanchez-Serrano, J. J. (1996) J. Biol. Chem. 271, 21012–2101920. Heitz, T., Bergey, D. R., and Ryan, C. A. (1997) Plant Physiol. 114, 1085–1093
21. Bell, E., Creelman, R. A., and Mullet, J. E. (1995) Proc. Natl. Acad. Sci.U. S. A. 92, 8675–8679
22. Rance, I., Fournier, J., and Esquerre-Tugaye, M. T. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 6554 – 6559
23. Royo, J., Leon, J., Vancanneyt, G., Albar, J. P., Rosahl, S., Ortego, F.,Castanera, P., and Sanchez-Serrano, J. J. (1999) Proc. Natl. Acad. Sci.U. S. A. 96, 1146–1151
24. Keil, M., Sanchez-Serrano, J. J., and Willmitzer, L. (1989) EMBO J. 8,1323–1330
25. Bevan, M. (1984) Nucleic Acids Res. 12, 8711–872126. Dammann, C., Rojo, E., and Sanchez-Serrano, J. J. (1997) Plant J. 11, 773–78227. Griffiths, G., Leverentz, M., Silkowski, H., Gill, N., and Sanchez-Serrano, J. J.
(2000) J. Exp. Bot. 51, 1363–137028. Christie, W. W. (1982) Lipid Analysis, 2nd Ed., Pergamon Press, Oxford29. Conconi, A., Miquel, M., Browse, J. A., and Ryan, C. A. (1996) Plant Physiol.
111, 797– 80330. Narvaez-Vasquez, J., Florin-Christensen, J., and Ryan. C. A. (1999) Plant Cell
11, 2249–226031. Ohlrogge, J and Browse, J. (1995) Plant Cell 7, 957–97032. Weber, H., Vick, B. A., and Farmer, E. E. (1997) Proc. Natl. Acad. Sci. U. S. A.
94, 10473–1047833. Blee, E. (1998) Prog. Lipid Res. 37, 33–7234. Gardner, H. W., Grove, M. J., and Salch, Y. (1996) J. Agric. Food Chem. 44,
882– 88635. Hamberg, M., and Gardner, H. W. (1992) Biochim. Biophys. Acta 1165, 1–1836. Gundlach, H., and Zenk, M. H. (1998) Phytochemistry 47, 527–53737. Vancanneyt, G., Sanz, C., Farmaki, T., Paneque, M., Ortego, F., Castanera, P.,
and Sanchez-Serrano, J. J. (2001) Proc. Natl. Acad. Sci. U. S. A. 98,8139 – 8144
38. Kunz, C., Schob, H., Stam, M., Kooter, J. M., and Meins, F. (1996) Plant. J. 10,437– 450
39. Benfey, P. N., and Chua, N.-H. (1990) Science 250, 959 –96640. Neuhuber, F., Park, Y.-D., Matzke, A. J. M., and Matzke, M. A. (1994) Mol.
Gen. Genet. 244, 230–24141. McKenzie, M. J., Mett, V., Stewart Reynolds, P. H., and Jameson, P. E. (1998)
Plant Physiol. 116, 969 –97742. Ruegger, M., Dewey, E., Hobbie, L., Brown, D., Bernascono, P., Turner, J.,
Muday, G., and Estelle, M. (1997) Plant Cell 9, 745–75743. Clouse, S. D., Langford, M., and McMorris, T. C. (1996) Plant Physiol. 111,
671– 678
44. Napoli, C. A., Beveridge, C. A., and Snowden, K. C. (1999) Curr. Top. Dev. Biol.44, 127–169
45. Stam, M., de Bruin, R., Kenter, S., van der Horn, R. A. L., Soppe, W. J.,Bentsink, L., and Koornneef, M. (1999) Development 126, 4763– 4770
46. Blee, E., and Joyard, J. (1996) Plant Physiol. 110, 445–45447. Kohlmann, M., Bachmann, A., Weichert, H., Kolbe, A., Balkenholt, T.,
Wasternack, C., and Feussner, I. (1999) Eur. J Biochem. 260, 885– 89548. Banas, A., Johansson, I., and Stymne, S. (1992) Plant Sci. 84, 137–14449. Hamberg, M. (1999) Lipids 34, 1131–114250. Orozco-Cardenas, M., Narvaez-Vasquez, J., and Ryan, C. A. (2001) Plant Cell
13, 179–191
LOX H1-depleted Transgenic Potato Plants 423