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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

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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 harvested 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-

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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.

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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.


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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.


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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


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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


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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.

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