A Knock-Out Mutation In Allene Oxide Synthase Results In Male Sterility and Defective Wound Signal...

A knock-out mutation in allene oxide synthase results inmale sterility and defective wound signal transduction inArabidopsis due to a block in jasmonic acid biosynthesis

Joon-Hyun Park1,2, Rayko Halitschke3, Ho Bang Kim4, Ian T. Baldwin3, Kenneth A. Feldmann1,2,* and ReneÂ Feyereisen5

1Ceres Inc., 3007 Malibu Canyon Road, Malibu, CA 90265, USA, 2Department of Plant Sciences, University of Arizona,

Tucson, AZ 85721, USA, 3Department of Molecular Ecology, Max Planck Institute for Chemical Ecology, Winzerlaer

Str. 10, D-07745 Jena, Germany, 4School of Biological Sciences, Seoul National University, Seoul 151-742, Korea, and5INRA Centre de Recherches d'Antibes, 1382 Route de Biot, 06560 Valbonne, France

Received 25 January 2002; revised 6 March 2002; accepted 13 March 2002*For correspondence (fax: +1 310 317 8998; email: [email protected])

Summary

Recent studies on jasmonic acid (JA) biosynthetic mutants have shown that jasmonates play essential

roles in pollen maturation and dehiscence and wound-induced defence against biotic attacks. To better

understand the biosynthetic mechanisms of this essential plant hormone, we isolated an Arabidopsis

knock-out mutant defective in the JA biosynthetic gene CYP74A (allene oxide synthase, AOS) using

reverse genetics screening methods. This enzyme catalyses dehydration of the hydroperoxide to an

unstable allene oxide in the JA biosynthetic pathway. Endogenous JA levels, which increase 100-fold 1 h

after wounding in wild-type plants, do not increase after wounding in the aos mutant. In addition, the

mutant showed severe male sterility due to defects in anther and pollen development. The male-sterile

phenotype was completely rescued by exogenous application of methyl jasomonate and by

complementation with constitutive expression of the AOS gene. RT±PCR analysis showed that the

induction of transcripts for vegetative storage protein and lipoxygenase genes, previously shown to be

inducible by wound and jasmonate application in the wild-type, was absent in the aos mutant. In

transgenic plants constitutively expressing AOS, wound-induced JA levels were 50±100% higher

compared to wild-type plants. Taken together with JA de®ciency in the aos mutant, our results show

that AOS is critical for the biosynthesis of all biologically active jasmonates. Our results also suggest

that AOS expression is limiting JA levels in wounded plants, but that the AOS hydroperoxide substrate

levels, controlled by upstream enzymes (lipoxygenase and phospholipase), determine JA levels in

unwounded plants.

Keywords: allene oxide synthase, methyl jasmonate, wounding, cytochrome P450, CYP74A

Introduction

Jasmonates are cyclopentanone derivatives that originate

biosynthetically from linolenic acid (LA) via an inducible

octadecanoid pathway consisting of several enzymatic

steps. Jasmonic acid (JA), the end-product of this path-

way, its methyl ester, methyl jasmonate (MeJA), and

certain L-amino acid conjugates, glucose esters and

hydroxylated forms are all found in plants (Sembdner

and Parthier, 1993). Generally, among these jasmonate

derivatives, JA and MeJA are referred to as `jasmonates'

and are recognized as the most potent plant growth

regulators, acting as signals for various developmentally

and environmentally induced changes in gene expression

(Baldwin et al., 1997; Creelman and Mullet, 1997;

Wasternack and Parthier, 1997). A recent report shows

that cyclopentenone precursors of JA can also regulate

wound induction of genes (Stintzi et al., 2001).

The roles of jasmonates in response to biotic stress,

such as insect and fungal attack (Bennett and Wallsgrove,

1994; McConn et al., 1997; Ozawa et al., 2000), and abiotic

stress, such as mechanical wounding, have been well

documented (Baldwin et al., 1997; Creelman and Mullet,

1997; Reymond et al., 2000; Richard et al., 2000) and have

The Plant Journal (2002) 31(1), 1±12

ã 2002 Blackwell Science Ltd 1

implications for biology and agriculture. These JA-medi-

ated pathogen and wound signal transduction pathways

are co-regulated by interactions with other phytohor-

mones (Lee et al., 1996; Thomma et al., 1999; van Wees

et al., 2000; Winz and Baldwin, 2001).

LA, a trienoic fatty acid, serves as the substrate for the

biosynthesis of JA (McConn and Browse, 1996). The early

steps convert LA to 12-oxo-phytodienoic acid (OPDA) by

means of three enzymes located in the chloroplast,

lipoxygenase (LOX), allene oxide synthase (AOS) and

allene oxide cyclase (AOC; Figure 1). OPDA is reduced by

a putative peroxisome associated enzyme, 12-oxo-phyto-

dienoic acid reductase (OPR), whose product undergoes

three cycles of b-oxidation, presumably in the peroxisome,

to generate JA (Stintzi and Browse, 2000).

McConn and Browse (1996) ®rst showed that LA was

required for male fertility in Arabidopsis. They created a

triple mutant (fad3-2/fad7-2/fad8), lacking hexadecatrienoic

acids (16:3) and LA (18:3), and showed that the male

sterility in the mutant could be rescued by exogenous

application of JA. In recent years, mutants with lesions in

various JA biosynthetic enzymes have also been shown to

be defective in pollen maturation and release (Farmer

et al., 1998; Ishiguro et al., 2001; Sanders et al., 2000; Stintzi

and Browse, 2000; Zhao and Ma, 2000). Ishiguro et al.

(2001) identi®ed a T-DNA-tagged male-sterile mutant

(dad1) in which the fertility phenotype could be recovered

with exogenous application of LA or JA. The cloned gene

shared homology with lipases and was shown to hydro-

lyse phospholipids. Over-expression of DAD1 resulted in

plants that tended to be yellow±green presumably because

of excess hydrolysis of phospholipids in the chloroplast

membranes, damaging the chloroplasts. They hypothe-

sized that DAD1 is the ®rst committed enzyme in JA

synthesis as it catalyses the release of free LA from cellular

lipids (Figure 1). A knock-out mutant of an enzyme further

down the JA pathway, 12-oxophytodienoic acid-10, 11-

reductase 3 (encoded by the OPR3/DDE1 gene; Sanders

Figure 1. The jasmonate biosyntheticpathway.Linolenic acid (18:3), which is released frommembrane phospholipid by a lipolyticenzyme (DAD1), is converted to the alleneoxide (12,13-epoxy-octadecatrienoic acid) bya lipoxygenase (LOX) and allene oxidesynthase (AOS), a member of the cyto-chrome P450 enzyme family (CYP74A). Onecyclization, one reduction and three roundsof b-oxidation steps are needed to generatejasmonic acid, the end-product of thepathway from allene oxide. Alternatively,13-hydroperoxylinolenic acid is converted tocis-3-hexenal and Traumatin via HPL(CYP74B). LOX, lipoxygenase; DAD1,DEFECTIVE IN ANTHER DEHISCENCE 1;AOS, allene oxide synthase; HPL,hydroperoxy fatty acid lyase; cis-9-ODA,cis-9-oxo-dodecenoic acid; AOC, alleneoxide cyclase; OPR3, 12-oxo-phytodienoicacid-10,11-reductase 3; DDE1, DELAYEDDEHISCENCE 1.

2 Joon-Hyun Park et al.

ã Blackwell Science Ltd, The Plant Journal, (2002), 31, 1±12

et al., 2000; Stintzi and Browse, 2000) also showed severe

male sterility due to a defect in the timing of stomium

degeneration and release of pollen (Figure 1). The sto-

mium cells eventually degenerate and pollen is released in

dde1 plants but at a later stage of ¯ower development

(Sanders et al., 2000). This late stomium degeneration and

pollen dehiscence might be attributable to the other two

OPR isozymes (OPR1 and OPR2) whose expression was

detected predominantly in Arabidopsis roots and only at

low levels in ¯owers (Biesgen and Weiler, 1999), and/or by

an alternative pathway which may generate JA from 16:3

fatty acids (Farmer et al., 1998; Weber et al., 1997).

To better understand the role of jasmonate signalling in

pollen and anther development, we screened for AOS

knock-out mutants. As shown in Figure 1, AOS is of a

particular importance in jasmonate biosynthesis because

the substrate for AOS also serves as the substrate for

CYP74B in the C-6 volatile pathway (Figure 1). AOS

catalyses the dehydration of 13-(S)-hydroperoxylinolenic

acid to unstable 12,13-epoxylinolenic acid (allene oxide).

AOS is a cytochrome P450 enzyme (CYP74A) encoded by a

single-copy gene in Arabidopsis (Laudert et al., 1996).

CYP74A is a non-classical P450 in that it acts in the

absence of an NADPH-dependent P450 reductase and

oxygen (Schuler, 1996). This phenomenon occurs only in

members of the CYP74 family (Paquette et al., 2000; Werck-

Reichhart and Feyereisen, 2000). The Arabidopsis CYP74

family members are also unusual among P450s in that

they appear to contain chloroplast-targeting sequences

(Paquette et al., 2000).

In this study, we report the isolation of an AOS knock-out

mutant (which cannot make endogenous JA or its

cyclopentenone precursors) by a PCR-based reverse gen-

etics screening method (Winkler et al., 1998). Biochemical

analysis shows that the internal level of JA in the aos

mutant is below the detection limit of the assay even after

wound treatment. Characterization of the mutant pheno-

type indicated that the aos mutant has a severe male

sterility phenotype that did not recover during develop-

ment but was rescued by exogenous MeJA spraying. Also,

the aos mutant shows a defect in wound signal transduc-

tion for both Arabidopsis vegetative storage protein 2

(AtVSP2) and lipoxygenase 2 (AtLOX2) genes, which are

inducible by wound and JA treatments in wild-type plants.

Results

Screening and isolation of an Arabidopsis allene oxide

synthase (AOS) knock-out mutant

Allene oxide synthase (AOS), a cytochrome P450 enzyme

(CYP74A), is of particular importance in jasmonate bio-

synthesis because it commits 13-(S)-hydroperoxy linolenic

acid to the formation of jasmonates by catalysing the

dehydration of this substrate to 12,13-epoxy-linolenic acid

(allene oxide) (Figure 1). In Arabidopsis, only one copy of

the AOS gene is present, located on chromosome 5, and

alternative enzymes have not been reported to catalyse

this step (Laudert et al., 1996).

Using a PCR-based reverse genetics screening method

with CYP74A- and T-DNA border-speci®c primers on DNA

from 72,750 T-DNA insertion lines, a knock-out mutant for

CYP74A was isolated from the TJ lines (TJ1180; see

Experimental procedures). The T-DNA in these transfor-

mants confers kanamycin-resistance (KanR). Four kanamy-

cin-resistant seedlings from this pool contained the

appropriate PCR product when AOS- and border-speci®c

primers were used. PCR using two AOS-speci®c primers

con®rmed that three of the siblings were heterozygous for

the insert and one (TJ1180-1) was homozygous (data not

shown). TJ1180-1 displayed complete sterility. Figure 2

shows the AOS gene structure based on genomic and

cDNA sequences and relative locations and directions of

primers used to amplify gene-speci®c (CYP74A1F and

CYP74A1R) and T-DNA ¯anking region-speci®c bands

(TJRB and CYP74A1R). Analysis of the AOS ¯anking

sequence in TJ1180-1 showed that the T-DNA insertion

point was 98 bp upstream of the invariant cysteine residue

within the heme binding domain, which is highly con-

Figure 2. Schematic map of the AOS gene.The AOS gene and ¯anking genomic regions are designated by the open box and bold lines, respectively. The haem binding domain of AOS is indicatedby a closed box (Heme). The open triangle shows the T-DNA position in the exon region of AOS. The CYP74A1 gene contains no introns. The arrowsshow the locations and directions of primers used to screen for an aos mutant (CYP74A1F, CYP74A1R and TJRB) and to clone the AOS exon region(CYP74A1EF and CYP74A1ER).

A mutation in AOS causes male sterility 3


served and crucial for the functionality of cytochrome P450

proteins (data not shown).

TJ1180-1 was back-crossed to the wild-type and F1 and

F2 progeny were analysed for segregation of the sterility

and KanR phenotypes. All of the F1 progeny (n = 15) were

fertile as expected for a recessive mutation. The F2

progeny segregated 3:1 (401 KanR:127 KanS), indicating a

single T-DNA insertion with a functional kanamycin resist-

ance marker. The linkage of the sterile phenotype with the

kanamycin resistance marker representing the T-DNA

insertion was analysed in 72 F2 segregating progeny.

PCR analyses of 19 sterile F2 plants using AOS- and

border-speci®c primers showed that all were homozygous

for the aos T-DNA knock-out allele. Of 53 fertile plants, PCR

analyses showed that 36 were heterozygous for the

insertion while 17 plants lacked an insert. As such, the

segregation ratio of the F2 progeny was an acceptable

1:2:1 (wild-type:heterozygous:mutant; c2 = 0.11). These

combined results show tight linkage of the sterility

phenotype with the T-DNA insertion and suggest that the

sterility phenotype is caused by a knock-out of AOS.

Male sterility in the aos mutant is rescued by MeJA

treatment

The adult phenotype of an aos mutant plant is shown in

Figure 3. aos mutants show no overt developmental

defects, except for sterility. Following fertilization, siliques

of wild-type plants elongated and the ¯oral organs

senesced within a week (Figure 3, left). In the aos mutant,

however, the ¯oral organs senesced normally but the

siliques remained small, producing no seeds (Figure 3,

right).

Figure 4(a) shows open ¯owers of the wild-type and

aos mutant; petals and sepals were cut away to show the

pistil and anthers. In wild-type plants, anthers developed

fully and pollen dehisced onto the stigmatic papilla

(Figure 4a, left). The fertilized pistil elongated to generate

a silique containing the developing seeds. Following

fertilization, other ¯oral organs, such as sepals, petals

and anthers, senesced and fell away from the silique

(Figure 4b, left). In contrast, in the aos mutant, stamen

elongation and anther development were stalled at the

¯ower opening stage (Smyth et al., 1990) and the pollen

failed to dehisce (Figure 4a, right). Other processes,

including ¯oral development and ¯oral organ senescence,

were similar to wild-type (data not shown). Anther

development in aos mutants was completely blocked and

failed to recover (Figure 4a, right; Figure 4b). To test

whether a loss of JA biosynthesis was responsible for

the sterility, we tried to rescue the male-sterile phenotype

in the aos mutant by application of LA, an initial substrate

and starting material in jasmonate biosynthesis, OPDA, an

intermediate substrate, and MeJA, a methyl ester of

jasmonic acid.

Figure 4(b) shows a male-sterile aos mutant silique and

an aos mutant silique rescued to wild-type by application

of OPDA and MeJA. Two days after OPDA and MeJA

spraying, aos mutant siliques started to elongate and

reached wild-type silique size in a week, while LA-sprayed

siliques did not respond. Seeds of OPDA and MeJA-

rescued aos plants were collected and sown. All progeny

showed the male-sterile phenotype, con®rming that they

were selfed progeny of the aos mutant (data not shown).

These results show that MeJA can rescue the aos male-

sterile phenotype.

Constitutive AOS expression complements the male-

sterile phenotype of aos mutants

Co-segregation of the T-DNA insert with male sterility and

chemical complementation of the male-sterile phenotype

in aos mutants using MeJA provided strong evidence that

male sterility in the aos mutant is caused by a disruption of

AOS. However, to remove any possibility that the male-

sterile phenotype was caused by an unknown mutation, it

was important to show that expression of AOS could

complement the aos mutant phenotype. For functional

complementation of the aos mutation, we generated

transgenic lines (AOS-OE) carrying AOS under the control

of the cauli¯ower mosaic virus (CaMV) 35S promoter in

wild-type plants (see Experimental procedures).

To introduce the constitutive AOS construct into aos

mutants, AOS-OE pollen was used to fertilize aos mutants.

Twelve F1 plants, resulting from crosses with three

independent over-expression lines, were selfed to obtain

F2 segregating families. Six F2 progeny for each F1 parent

that were resistant to both antibiotics (kanamycin and

hygromycin) were selected and transferred to soil. The

genotypes of the transferred plants were tested by PCR

using speci®c primer sets for aos, AOS and the constitutive

AOS expression construct, and male sterility was exam-

ined at the ¯owering stage. All 72 F2 plants showed

normal fertility and silique elongation (Figure 5a). To

identify genotypes of selected F2 plants, sets of primers

were used to amplify the wild-type AOS-, T-DNA-inserted

aos- and constitutively expressed AOS-speci®c bands.

Figure 5(b) shows their orientation and relative locations

on a schematic drawing of the aos and constitutive AOS

gene structure. Genotyping results indicated that 26 out of

72 plants were homozygous for the aos mutation and

contained an AOS-OE construct (Figure 5c). From these

results, it is clear that the male sterility phenotype in

the aos mutant is completely rescued by the AOS-OE

construct.



Figure 3. Phenotypes of aos mutants.Wild-type (left) and Arabidopsis aos mutant (right) plants 30 days after germination. The third and fourth siliques of wild-type (arrowheads) show normaldevelopment. The ¯owers of aos (arrowheads) fail to make siliques. The inset pictures show the ®rst three siliques from the primary in¯orescence of awild-type plant (left) and aos mutant (right) 1 week after ¯ower opening. Scale bars, 1 cm.

Figure 4. Recovered fertility of aos by exogenous application of methyl jasmonate.(a) Opened ¯owers of wild-type (left) and aos mutant (right). Anthers of the wild-type dehisced and pollen adhered to the stigmatic papilla (arrowhead).The aos anthers do not fully elongate and fail to dehisce (arrowhead). (b) From left, fully developed siliques of untreated wild-type, aos mutant, and aossiliques treated with LA, OPDA or MeJA, 2 weeks after ¯ower opening. Scale bars (a) 1 mm; (b) 5 mm.



The AOS knock-out mutation blocks wound signal

transduction as well as JA biosynthesis

To address whether JA biosynthesis is blocked in the aos

mutant, we measured internal jasmonate levels in wild-

type Col-6 and aos mutant plants after wounding. In the

absence of wounding or pathogen attack, the level of JAs

is barely detectable in Arabidopsis (Creelman et al., 1992;

Kramell et al., 2000; Weichert et al., 2000). After wound

treatment, jasmonate levels were dramatically increased

and reached a peak 1 h after wounding (Figure 6a).

Internal jasmonate concentration returned to normal levels

Figure 5. Rescue of the male sterility phenotype in the aos mutant with the constitutive AOS construct.(a) From left, primary in¯orescence and siliques of wild-type, wild-type carrying the AOS-OE construct, aos mutant, and a aos mutant carrying the AOS-OEconstruct 35 days after germination. Scale bar, 1 cm. (b) The AOS gene and ¯anking genomic regions (top) and the AOS over-expression construct(AOS-OE) and ¯anking regions originating from pCAMBIA1300 (bottom) are designated by open boxes and bold lines, respectively. The closed boxesshow the 35S promoter (P) and 35S poly(A) signal sequences (T) in the over-expression construct. The open triangle shows the T-DNA insertion in theexon region of AOS (not drawn to scale). The arrows show locations and directions of primers used to identify genotypes of F2 progeny between aos andAOS-OE. The right border primer of the TJ line and a gene-speci®c primer (top; TJRB and CYP74A1R) were used to amplify the aos gene-speci®c band. Apair of gene-speci®c primers was used to generate the wild-type AOS gene-speci®c band (centre; CYP74A1F2 and CYP74A1R). One gene-speci®c primerand one plant binary vector (pCAMBIA1300) speci®c primer were used to amplify the over-expression construct speci®c bands (bottom; CYP74A1F2 andpCAM1300F). (c) Genotyping results. From left to right, wild-type, wild-type carrying AOS-OE, aos mutant, and an aos mutant carrying the AOS-OEconstruct.



1 day after wounding. In contrast, aos mutants showed no

induction of JA after wounding, and internal JA levels

were not signi®cantly different from unwounded wild-type

plants (P values > 0.93). The induction kinetics of JA

accumulation indicate that JA biosynthesis is completely

blocked by the aos mutation in Arabidopsis.

Jasmonic acid and methyl jasmonate have been

reported to perform roles in wound responses in plants

(Farmer and Ryan, 1990; Gundlach et al., 1992), as well as

in developmental processes (Creelman and Mullet, 1997;

Sembdner and Parthier, 1993). To investigate wound

responses in aos mutants, expression levels of AOS and

two wound- and jasmonate-responsive genes (Bell and

Mullet, 1993; Laudert and Weiler, 1998; Utsugi et al., 1998),

A. thaliana vegetative storage protein 2 (AtVSP2) and

lipoxygenase 2 (AtLOX2), were examined by RT±PCR

after wound treatment (Figure 6b). The expression level

of AOS was maximally induced 2 h after treatment and

then decreased gradually to near normal levels in wild-

type, consistent with the kinetics of jasmonate synthesis.

In aos mutants, AOS transcripts were not detected even in

wound-induced conditions, indicating that the T-DNA

insertion prevents the accumulation of any transcript

from this gene (Figure 6b). The two other wound-inducible

genes, AtVSP2 and AtLOX2, reached maximum transcript

levels 4 h after treatment in wild-type and rapidly

Figure 6. Induction of jasmonates and jasmonate-inducible genes by wound treatment.(a) Kinetics of wound-induced JA levels (mean 6 SE of four replicates) of wild-type (Col-6, s) and aos (d) Arabidopsis plants. (b) Ampli®cation by RT±PCRof AtLOX2, AOS and AtVSP2 genes after wound treatment. Total RNA was isolated from wounded leaves of wild-type plants and aos mutants at 2, 4 and8 h after wounding. As a control, the Arabidopsis ACTIN-2 gene was ampli®ed.



decreased to normal expression levels. Interestingly,

expression levels of AtVSP2 and AtLOX2 in aos mutants

were not affected by wound treatment, indicating that the

wound signal to AtLOX2 and AtVSP2 was severed by the

aos mutation (Figure 6b).

It is clear that the biosynthesis of all biologically active

jasmonates was blocked by the aos mutation and that

wound signal transduction to AtLOX2 and AtVSP2 was

interrupted by blocking JA production.

Constitutive expression of AOS causes an increase in

wound-induced JA levels

Three transgenic lines expressing AOS under the control

of the CAMV 35S promoter were analysed for their JA

levels and for the relative AOS, AtLOX2 and AtVSP2

transcript levels as was done for the aos mutant plants.

JA levels in healthy AOS-OE plants were similar to those in

wild-type plants, but 1 h after wounding, JA reached levels

higher than those observed in wounded wild-type plants

(Figure 7a). In line AOS-OE 4, wound-induced JA levels

increased twofold to over 2.2 mg g±1 FW, thus con®rming

the results of Laudert et al. (2000). RT±PCR experiments

showed that AOS, AtLOX2 and AtVSP2 were induced

faster in the AOS-OE lines than in wild-type plants.

Transcript levels in unwounded AOS-OE plants also

appeared higher than in their wild-type counterparts,

perhaps indicating an exquisite sensitivity of these plants

to JA, but this point deserves a more thorough analysis.

Discussion

The structure and biosynthesis of JA have intrigued plant

biologists because of parallels to eicosanoid second

messengers that are central to in¯ammatory responses

and other physiological processes in mammals (Creelman

et al., 1992). Thus, a pathway that is initiated by lipoxy-

genase synthesizes JA from LA, a precursor that is

presumably released from chloroplast membrane lipids

by the action of a phospholipase. Cyclization and b-

oxidation of the lipoxygenase product, 13-(S)-hydroperox-

ylinolenic acid, result in JA having a structure analogous in

some respects to the prostaglandin E series of eicosanoids

(Vick and Zimmerman, 1983).

In the JA-speci®c biosynthetic pathway (Figure 1), a

cytochrome P450 gene CYP74A (allene oxide synthase) is

of particular importance because it catalyses dehydration

of 13-(S)-hydroperoxylinolenic acid to 12,13-epoxy-linole-

nic acid (allene oxide), the ®rst committed step in JA

synthesis rather than synthesis of C-6 volatiles. At the

molecular level, the AOS gene is found as a single-copy

gene on chromosome 5.

To study biosynthetic mechanisms and wound signal

transduction of this essential plant hormone, we isolated a

T-DNA knock-out mutant using reverse genetics screening

methods (Figure 2). We measured endogenous JA levels

in wild-type and aos mutant plants. As shown in

Figure 6(a), the basal level of JA in wild-type Arabidopsis

tissue was barely detectable at 12.7 6 2.5 ng g±1 FW

(mean 6 SE). The endogenous JA level soared to a

maximum 1 h after wound treatment to attain concentra-

tions approximately 100-fold higher than those of

unwounded plants. JA levels in the aos mutant were not

different from those of unwounded wild-type plants and

were not changed after wound treatment (Figure 6a).

Thus, the knock-out mutation of AOS blocks the jasmonate

biosynthetic pathway completely.

Jasmonates are essential for Arabidopsis male fertility

Recent studies on JA biosynthetic mutants have shown

that JAs play essential roles in pollen maturation and

dehiscence (Ishiguro et al., 2001; Sanders et al., 2000;

Stintzi and Browse, 2000). The aos mutant showed severe

male sterility due to defects in anther development

(Figure 3). To complement the sterility of the aos mutant,

we applied LA, 12-oxo-phytodienoic acid OPDA and MeJA,

Figure 7. JA content and expression levels of jasmonate inducible genesin AOS-OE lines.(a) Mean 6 SE JA levels of wild-type (Col-0) and AOS-OE Arabidopsisplants (OE-4, OE-7, OE-10); ®ve replicates per treatment). Samples weretaken 1 h after wound treatment (W) or from undamaged control plants(C). (b) Ampli®cation by RT±PCR of AtLOX2, AOS and AtVSP2 genes afterwound treatment. Total RNA was isolated from wounded leaves of wild-type plants and transgenic lines carrying the AOS-OE 7 construct at 0.5,1, 2 4, and 8 h after wounding. The Arabidopsis ACTIN-2 gene wasampli®ed as a control.



which are a substrate, an intermediate and a methylated

form of the end-product in the JA biosynthetic pathway,

respectively. The male-sterile phenotype was completely

rescued by exogenous OPDA and MeJA, while LA failed to

produce a response (Figure 4). The male-sterile phenotype

of the aos mutant was complemented by constitutive AOS

expression under the control of the CaMV 35S promoter in

plants homozygous for the aos mutation and shown to

carry AOS-OE constructs. Thus AOS is necessary and

suf®cient to complement the sterility in the aos mutant.

Taken together, it is clear that blocking JA biosynthesis

caused the male sterility phenotype of the aos mutant.

This physiological evidence shows that AOS might be the

only enzyme to act on hydroperoxide substrates from both

the octadecanoid pathway and the hexadecanoid pathway

(proposed as another source of plant oxylipins; Farmer

et al., 1998). In the hexadecanoid pathway, 16:3 fatty acids

would be used as starting materials to produce JA-like

oxylipins through dinor-oxo-phytodienoic acid (dnOPDA),

leading to JA.

AOS acts as a primary wound signal modulator

It has been shown repeatedly that wounding leads to an

accumulation of JA and to substantial increases in AOS

transcript levels and enzyme activity (Laudert and Weiler,

1998). To assess the effect of the aos knock-out mutation in

wound signal transduction, transcript levels of wound-

inducible genes such as lipoxygenase 2 (AtLOX2) and

vegetative storage protein 2 (AtVSP2) were measured by

RT±PCR analysis after wounding. The internal level of JA

was dramatically induced in 30 min, reached a maximum

at 1 h, and then returned to normal levels relatively slowly

when compared to the induction kinetics (Figure 6a).

RT±PCR analysis showed that transcripts of AtLOX2 and

AtVSP2 were not induced and remained at the untreated

level in the aos mutant after wound treatment (Figure 6b).

This indicated that wound signal transduction to AtLOX2

and AtVSP2 was not generated or was blocked by the lack

of endogenous jasmonates. Also, the expression of AOS

reached a maximum level within 2 h after wound treat-

ment in wild-type plants (Figure 6b). Meanwhile, the

induction kinetics of AtLOX2, whose product acts

upstream of AOS in JA biosynthesis, shows a slower

induction and much faster down-regulation kinetics than

AOS in wild-type plants.

To examine further the function of AOS in wound signal

transduction in Arabidopsis, AOS-OE plants were

wounded and JA levels and wound-induced gene expres-

sion were measured (Figure 7). Con®rming the results of

another study (Laudert et al., 2000), JA levels were not

changed in unwounded plants, but were increased by

wounding, well over the levels found in wounded wild-

type plants. Interestingly AOS, AtLOX2 and especially

AtVSP2 showed much faster induction in the AOS-OE

plants than in the wild-type. In the AOS-OE line, AtVSP2

induction reached a maximum around 0.5 h after wound-

ing, compared to 4 h in the wild-type. These results

provide clear evidence that the JA-dependent wound

signal transduction pathway is highly potentiated by

constitutive expression of AOS in Arabidopsis. They also

suggest that AOS expression is limiting JA levels in

wounded plants, as higher AOS expression leads to higher

JA levels. JA levels in unwounded plants are determined

by hydroperoxide substrate levels for AOS, and not by

AOS activity, as constitutive expression of AOS does not

increase basal JA levels. In contrast to our results, Harms

et al. (1995) showed elevated JA levels in unwounded

potato tubers over-expressing ¯ax AOS. This contradiction

might be due to the differences in substrate availability

between the two plant systems. The levels of the substrate,

fatty acid hydroperoxide, are controlled by enzymes

upstream of AOS (lipoxygenase and phospholipase). The

regulation of these genes and the resulting increased

availability of the product, the substrate of AOS, might

affect the basal level of JA. These results suggest that the

expression of these genes, and the substrate availability, is

more tightly regulated in Arabidopsis. As shown by Harms

et al. (1995), prolonged high endogenous JA levels in the

AOS over-expressing potatoes possibly cause a lack of

wound- and water-stress responses in JA signal transduc-

tion. This self-regulatory mechanism could prevent the

production of potentially toxic free fatty acids and fatty

acid hydroperoxides, as well as the malfunction of wound

signal transduction, in unwounded plants.

In conclusion, our results support the hypothesis that

AOS is critical in the biosynthesis of all biologically active

jasmonates and is a major regulator of the wound signal

transduction pathway in Arabidopsis.

Experimental procedures

Mutant isolation

To isolate Arabidopsis thaliana aos mutants, we screened T-DNAmutagenized Arabidopsis lines generated by Tom Jack (TJ;ecotype Col-6; 6000 lines), Ken Feldmann (KF; ecotypeWassilewskija (Ws-2); 6000 lines) and the Arabidopsis knock-outfacility (AKF; http://www.biotech.wisc.edu/Arabidopsis/ ecotypeWs-2; 60 750 lines) by reverse genetics using combinations ofgene- and T-DNA border-speci®c primers (Winkler et al., 1998).

Based on the sequence of wild-type genomic DNA, a pair ofgene-speci®c primers (CYP74A1F, 5¢-AACATATGCTCAAGGGAT-GGAGCTAAAAG-3¢ and CYP74A1R, 5¢-CGAACATGTAGAGCAG-CAACTGATTATACA-3¢) were designed to screen for mutants.TJLB (5¢-CTTTCGCCTATAAATACGACGGATCGTA-3¢) and TJRB(5¢-CGGGCCTAACTTTTGGTGTGATGATGCT-3¢) primers for TJlines, KFLB (5¢-GATGCACTCGAAATCAGCCAATTTTAGAC-3¢) andKFRB (5¢-TCCTTCAATCGTTGCGGTTCTGTCAGTTC-3¢) primers forKF lines, and JL202 (5¢-CATTTTATAATAACGCTGCGGACATCTAC-3¢) and JL270 (5¢-TTTCTCCATATTGACCATCATACTCATTG-3¢)



primers for AKF lines were used as T-DNA border-speci®cprimers. To amplify the regions ¯anking the T-DNA insertion,the primers CYP74A1F and CYP74A1R were used. After primaryscreening of super-pools by PCR, using combinations of thesegene- and border-speci®c primers, ampli®ed bands were blottedonto a nylon membrane (Roche, Indianapolis, IN, USA) andprobed by a digoxygenin (DIG)-labelled AOS gene-speci®c probeto identify positive super-pools. The preparation of the DIG-labelled probe and Southern blot analysis were performed usinga DIG DNA Labeling and Detection Kit (Roche). After a secondround of screening the corresponding sub-pools, ampli®ed DNAfragments were puri®ed using Qiaquick columns (Qiagen Inc.,Germantown, MD, USA) and sequenced with the border primer,to identify the T-DNA insertion site in the AOS gene. Thisscreening procedure yielded an aos knock-out mutant in TJ line1180 which is available through the Arabidopsis BiologicalResource Center (ABRC) seed catalogue (http://aims.cps.msu.edu/aims).

Plant growth conditions and wound treatment

Arabidopsis wild-type (Col-6) and aos mutant seeds were sown in7 cm round pots containing Metro-Mix 350 (Scotts, USA) andcold-treated at 4°C for 3 days. After cold treatment, plants wereincubated in a controlled growth room under long-day conditions(16 h of light (with a minimum intensity of 100 mmol m±2 sec±1) at22°C/8 h of dark at 20°C). For wound treatments, all but the 1stand 2nd rosette leaves of 4-week-old-plants were crushed severaltimes across the mid-vein with laminated forceps, which effect-ively wounded 40±50% of the leaf area. Plants were incubated for0, 2, 4 and 8 h, after which the leaves were harvested andimmediately frozen in liquid nitrogen. The 0 h time point indicatesharvest of leaves from unwounded control plants. For over-expression lines, wounded leaves were harvested and frozen at 0,0.5, 1, 2, 4 and 8 h after wound treatment.

Jasmonate measurement and chemical treatments

The four youngest fully developed leaves of 4-week-old plants(four per treatment) were wounded with a pattern wheel produ-cing three rows of puncture wounds on each leaf half. The treatedleaves were harvested at the indicated time points (0.5, 1, 2, 4, 8,24 and 48 h) and immediately frozen in liquid nitrogen. The 0 htime point indicates harvest of leaves from unwounded controlplants. The samples were analysed by GC±MS after addition of172 ng of 13C1,2-JA as an internal standard and sample prepar-ation as described by Schittko et al. (2000) with the followingmodi®cations of the extraction procedure. Leaf samples werehomogenized in the extraction buffer by utilizing the FastPrepextraction system FP120 (Savant Instruments, Holbrook, NewYork State, USA). Tissue was homogenized with a reciprocatingshaker at 6.0 m sec±1 for 90 sec in extraction tubes containing900 mg of lysing matrix (BIO 101, Vista, California, USA).

To test for recovery of male fertility in aos mutants, 4-week oldplants, grown under long-day conditions, were treated with either1% linolenic acid (LA; Cayman Chemical, Ann Arbor, MI, USA),2 mM MeJA or 1 mM oxo-phytodienoic acid (OPDA; CaymanChemical). Floral buds were dipped into OPDA supplementedwith 0.01% Triton X-100 (Sigma-Aldrich, St Louis, MO, USA) whileLA and MeJA were sprayed directly on ¯ower buds. The plantswere treated twice per day for two days and monitored for siliqueelongation over the next two weeks.

Extraction of RNA and RT±PCR analysis

Total RNA of frozen tissues was extracted with Trizol (Sigma-Aldrich). After RNAase-free DNAase treatment, 1 mg of total RNAwas used to create ®rst-strand cDNA using SuperScript IIRnase H± reverse transcriptase (Invitrogen, Carlsbad, CA, USA).

To analyse wound-induced expression levels of Arabidopsisthaliana vegetative storage protein 2 (AtVSP2), lipoxygenase 2(AtLOX2) and AOS genes, RT±PCR analyses were performedusing the ®rst-strand cDNAs as templates. CYP74A1EF (5¢-CCA-TATTCTAGATCACAACACTCGCCACTG-3¢)/CYP74A1ER (5¢-CAG-ATTCTAGAACATTTTCTCAAAATTCACG-3¢), ATVSP2F (5¢-ACA-TCACGAATTCAACAATAAACCATACCAT-3¢)/ATVSP2R (5¢-GGA-GAATTCGATGAAGATAGATTCTTAAGAA-3¢), and ATLOX2F (5¢-TTGGCTGAGGAAGATAAGACCGCAGAACAT±3¢)/ATLOX2R (5¢-TCATTTTATCAAGAAGACAGAGATACAGAA-3¢) primer sets wereused as speci®c primers of AOS, AtVSP2 and AtLOX2 genes,respectively. The conditions for PCR ampli®cation were asfollows: denaturing at 95°C for 3 min and then 30 cycles at 94°Cfor 30 sec, 58°C for 30 sec, and 72°C for 2 min. As a positivecontrol, the Arabidopsis ACTIN-2 gene was ampli®ed under thesame conditions using the following two primers: ACT2F (5¢-ACATCACGAATTCAACAATAAACCATACCAT-3¢) and ACT2R (5¢-GGAGAATTCGATGAAGATAGATTCTTAAGAA-3¢).

Isolation of AOS over-expression lines

The wild-type Arabidopsis AOS gene was ampli®ed withCYP74A1EF (5¢-CCATATTCTAGATCACAACACTCGCCACTG-3¢)and CYP74A1ER (5¢-CAGATTCTAGAACATTTTCTCAAAATTCACG-3¢) primers (underlined letters show modi®ed sequences forcloning by enzyme digestion). The ampli®ed AOS gene wasdigested with XbaI and cloned into XbaI-digested pUC19 vectors.The cloned Arabidopsis genes were sequenced with CYP74A1EFand CYP74A1ER. To complete sequencing of the cloned AOSgene, CYP74A1EF2 (5¢-GGAGCCTAAACACGAAAAGCTC-3¢) andCYP74A1ER2 (5¢-TTAACTCCATTTTCTCAATCGC-3¢) primers weredesigned. The cloned Arabidopsis AOS gene was isolated by SalIand XbaI double digestion and cloned into pRT101 vectorscontaining the 35S promoter and the polyadenylation signaldigested with XhoI and XbaI (ToÈ pfer et al., 1987). The over-expression construct including the 35S promoter and polyadeny-lation signal of the CaMV and the Arabidopsis AOS gene wasisolated by digestion with PstI and ligated into the PstI site ofAgrobacterium binary vector pCAMBIA1300 (AF234296). Theconstruct was transformed initially into Agrobacterium strainGV3101 by electroporation and then into the wild-typeArabidopsis ecotype Col-0 by the ¯oral dip method (Clough andBent, 1998).

Seeds collected from the transformed plants were surface-sterilized with 30% bleach and 0.01% Triton X-100 (Sigma) for10 min, and washed ®ve times with sterile distilled water. Aftersurface sterilization, seeds were plated on 0.8% agar-solidi®edmedium containing Murashige-Skoog (MS) salts (Invitrogen)supplemented with 2% sucrose and 50 mg l±1 hygromycin B(Invitrogen) and stored at 4°C for 3 days. After vernalization, theseeds were transferred to long-day conditions (16 h light [with aminimum intensity of 100 mmol m±2 sec±1] at 22°C/8 h dark at20°C). Ten independent hygromycin-resistant T1 seedlings weretransferred to soil and segregating T2 seeds were harvested. Toisolate homozygous lines for the over-expression construct, sixborder-speci®c primers (pCAMTAIL1L, 5¢-CAATACGAGGTC-GCCAACATCTTCTTCT-3¢; pCAMTAIL2L, 5¢-CAACTCTATCAG-AGCTTGGTTGACGGCA-3¢; pCAMTAIL3L, 5¢-CTGTGTAGAAGTA-



CTCGCCGATAGTGGAAA-3¢; pCAMTAIL1R, 5¢-CACTGGCCGTCG-TTTTACAACGTCGTGA-3¢; pCAMTAIL2R, 5¢-GCACCGATCGCC-CTTCCCAACAGTT-3¢; pCAMTAIL3R, 5¢-CAGATTGTCGTTTCC-CGCCTTCAGTTTA-3¢) were designed and ¯anking regions ofover-expression constructs were ampli®ed by thermal asymmet-ric interlaced (TAIL)±PCR (Liu et al., 1995). Based on sequences ofthe ¯anking region, speci®c primers were designed and thegenotypes of T2 families were examined. Three independenthomozygous lines were chosen for measurement of internal andwound-induced JA accumulation.

Complementation analysis

For complementation analysis, aos mutants were crossed to AOSover-expression lines. Three independent AOS over-expressionplants were used as pollen donors to cross to aos mutants. The 12F2 segregating families from these crosses were examined byPCR to identify genotypes of segregating F2 plants. Two AOS-speci®c primers, CYP74A1F2 (5¢-GTACGATCAAAGCCGTTGAAA-TTCCGAGTTTT-3¢) and CYP74A1R2 (5¢-TGGAACAAGAAAACAGA-TGGACTACACAGGT-3¢) and a pCAMBIA1300-speci®c primer,pCAM1300F (5¢-CTCATTAGGCACCCCAGGCTTTACACTT-3¢) weredesigned. Six kanamycin- and hygromycin-resistant plants foreach of the independent F2 families were selected and transferredto soil and grown under long-day conditions as described above.For genotyping of F2 plants, genomic DNA was isolated from onerosette leaf per plant with 500 ml of extraction buffer (0.2 M Tris±Cl, pH 9.0, 1% SDS, 25 mM EDTA, and 0.4 M LiCl). Plant statureand fertility (based on the formation of siliques) of F2 plants werescored.

Acknowledgements

This work was supported by the Human Frontiers ScienceProgram (grant RG0280/1999M). We also thank Sunghwa Choefor many useful discussions and Ed Kiegle for critical review ofthis manuscript.

References

Baldwin, I.T., Zhang, Z.-P., Diab, N., Ohnmeiss, T.E., McCloud,E.S., Lynds, G.Y. and Schmelz, E.A. (1997) Quanti®cation,correlations and manipulations of wound-induced changes injasmonic acid and nicotine in Nicotiana sylvestris. Planta, 201,397±404.

Bell, E. and Mullet, J.E. (1993) Characterization of an Arabidopsislipoxygenase gene responsive to methyl jasmonate andwounding. Plant Physiol. 103, 1133±1137.

Bennett, R.N. and Wallsgrove, R.M. (1994) Secondary metabolitesin plant defense mechanisms. New Phytol. 127, 617±633.

Biesgen, C. and Weiler, E.W. (1999) Structure and regulation ofOPR1 and OPR2, two closely related genes encoding 12-oxophytodienoic acid-10,11-reductases from Arabidopsisthaliana. Planta, 208, 155±165.

Clough, S.J. and Bent, A.F. (1998) Floral dip: a simpli®ed methodfor Agrobacterium-mediated transformation of Arabidopsisthaliana. Plant J. 16, 735±743.

Creelman, R.A. and Mullet, J.E. (1997) Biosynthesis and action ofjasmonates in plants. Annu. Rev. Plant Physiol. Plant Mol. Biol.48, 355±381.

Creelman, R.A., Tierney, M.L. and Mullet, J.E. (1992) Jasmonicacid/methyl jasmonate accumulate in wounded soybean

hypocotyls and modulate wound gene expression. Proc. NatlAcad. Sci. USA, 89, 4938±4941.

Farmer, E.E. and Ryan, C.A. (1990) Interplant communication:airborne methyl jasmonate induces synthesis of proteinaseinhibitors in plant leaves. Proc. Natl Acad. Sci. USA, 87, 7713±7716.

Farmer, E.E., Weber, H. and Vollenweider, S. (1998) Fatty acidsignaling in Arabidopsis. Planta, 206, 167±174.

Gundlach, H., Muller, M.J., Kutchan, T.M. and Zenk, M.H. (1992)Jasmonic acid is a signal transducer in elicitor-induced plantcell cultures. Proc. Natl Acad. Sci. USA, 89, 2389±2393.

Harms, K., Atzorn, R., Brash, A., KuÈ hn, H., Wasternack, C.,Willmitzer, L. and PenÄ a-CorteÂ s, H. (1995) Expression of a ¯axallene oxide synthase cDNA leads to increased endogenousjasmonic acid (JA) levels in transgenic potato but not to acorresponding activation of JA-responding genes. Plant Cell, 7,1645±1654.

Ishiguro, S., Kawai-Oda, A., Ueda, J., Nishida, I. and Okada, K.(2001) The DEFECTIVE IN ANTHER DEHISCENCE1 geneencodes a novel phospholipase A1 catalyzing the initial stepof jasmonic acid biosynthesis, which synchronizes pollenmaturation, anther dehiscence, and ¯ower opening inArabidopsis. Plant Cell, 13, 2191±2209.

Kramell, R., Miersch, O., Atzorn, R., Parthier, B. and Wasternack,C. (2000) Octadecanoid-derived alteration of gene expressionand the `oxylipin signature' in stressed barley leaves.Implications for different signaling pathways. Plant Physiol.123, 177±188.

Laudert, D. and Weiler, E.W. (1998) Allene oxide synthase: a majorcontrol point in Arabidopsis thaliana octadecanoid signalling.Plant J. 15, 675±684.

Laudert, D., Pfannschmidt, U., Lottspeich, F., Hollander-Czytko,H. and Weiler, E.W. (1996) Cloning, molecular and functionalcharacterization of Arabidopsis thaliana allene oxide synthase(CYP 74), the ®rst enzyme of the octadecanoid pathway tojasmonates. Plant Mol Biol. 31, 323±335.

Laudert, D., Schaller, F. and Weiler, E.W. (2000) TransgenicNicotiana tabacum and Arabidopsis thaliana plantsoverexpressing allene oxide synthase. Planta, 211, 163±165.

Lee, J., Parthier, B. and Lobler, M. (1996) Jasmonate signallingcan be uncoupled from abscisic acid signalling in barley:identi®cation of jasmonate-regulated transcripts which are notinduced by abscisic acid. Planta, 199, 625±632.

Liu, Y.G., Mitsukawa, N., Oosumi, T. and Whittier, R.F. (1995)Ef®cient isolation and mapping of Arabidopsis thaliana T-DNAinsert junctions by thermal asymmetric interlaced PCR. Plant J.8, 457±463.

McConn, M. and Browse, J. (1996) The critical requirement forlinolenic acid is pollen development, not photosynthesis, in anArabidopsis mutant. Plant Cell, 8, 403±416.

McConn, M., Creelman, R.A., Bell, E., Mullet, J.E. and Browse, J.(1997) Jasmonate is essential for insect defense in Arabidopsis.Proc. Natl Acad. Sci. USA, 94, 5473±5477.

Ozawa, R., Arimura, G., Takabayashi, J., Shimoda, T. andNishioka, T. (2000) Involvement of jasmonate- and salicylate-related signaling pathways for the production of speci®cherbivore-induced volatiles in plants. Plant Cell Physiol. 41,391±398.

Paquette, S.M., Bak, S. and Feyereisen, R. (2000) Intron±exonorganization and phylogeny in a large superfamily, theparalogous cytochrome P450 genes of Arabidopsis thaliana.DNA Cell Biol. 19, 307±317.

Reymond, P., Weber, H., Damond, M. and Farmer, E.E. (2000)Differential gene expression in response to mechanical



wounding and insect feeding in Arabidopsis. Plant Cell, 12,707±720.

Richard, S., Lapointe, G., Rutledge, R.G. and Seguin, A. (2000)Induction of chalcone synthase expression in white spruce bywounding and jasmonate. Plant Cell Physiol. 41, 982±987.

Sanders, P.M., Lee, P.Y., Biesgen, C., Boone, J.D., Beals, T.P.,Weiler, E.W. and Goldberg, R.B. (2000) The ArabidopsisDELAYED DEHISCENCE1 gene encodes an enzyme in thejasmonic acid synthesis pathway. Plant Cell, 12, 1041±1061.

Schittko, U., Preston, C.A. and Baldwin, I.T. (2000) Eating theevidence? Manduca sexta larvae cannot disrupt speci®cjasmonate induction in Nicotiana attenuata by rapidconsumption. Planta, 210, 343±346.

Schuler, M.A. (1996) Plant cytochrome P450 monooxygenases.Crit. Rev. Plant Sci. 15, 235±284.

Sembdner, G. and Parthier, B. (1993) Biochemistry, physiologicaland molecular actions of jasmonates. Annu. Rev. Plant Physiol.54, 328±332.

Smyth, D.R., Bowman, J.L. and Meyerowitz, E.M. (1990) Early¯ower development in Arabidopsis. Plant Cell, 2, 755±767.

Stintzi, A. and Browse, J. (2000) The Arabidopsis male-sterilemutant, opr3, lacks the 12-oxophytodienoic acid reductaserequired for jasmonate synthesis. Proc. Natl Acad. Sci. USA, 97,10625±10630.

Stintzi, A., Weber, H., Reymond, P., Browse, J. and Farmer, E.E.(2001) Plant defense in the absence of jasmonic acid: the role ofcyclopentenones. Proc. Natl Acad. Sci. USA, 98, 12837±12842.

Thomma, B.P., Eggermont, K., Tierens, K.F. and Broekaert, W.F.(1999) Requirement of functional ethylene-insensitive 2 genefor ef®cient resistance of Arabidopsis to infection by Botrytiscinerea. Plant Physiol. 121, 1093±1102.

ToÈ pfer, R., Matzeit, V., Gronenborn, B., Schell, J. and Steinbiss,H.-H. (1987) A set of plant expression vectors for transcriptionaland tanslational fusions. Nucl. Acids Res. 15, 5890.

Utsugi, S., Sakamoto, W., Murata, M. and Motoyoshi, F. (1998)

Arabidopsis thaliana vegetative storage protein (VSP) genes:gene organization and tissue-speci®c expression. Plant MolBiol. 38, 565±576.

Vick, B.A. and Zimmerman, D.C. (1983) The biosynthesis ofjasmonic acid: a physiological role for plant lipoxygenase.Biochem. Biophys. Res. Commun. 111, 470±477.

Wasternack, C. and Parthier, B. (1997) Jasmonate-signaled plantgene expression. Trends Plant Sci. 2, 302±307.

Weber, H., Vick, B.A. and Farmer, E.E. (1997) Dinor-oxo-phytodienoic acid: a new hexadecanoid signal in thejasmonate family. Proc. Natl Acad. Sci. USA, 94, 10473±10478.

van Wees, S.C., de Swart, E.A., van Pelt, J.A., van Loon, L.C. and

Pieterse, C.M. (2000) Enhancement of induced diseaseresistance by simultaneous activation of salicylate- andjasmonate-dependent defense pathways in Arabidopsisthaliana. Proc. Natl Acad. Sci. USA, 97, 8711±8716.

Weichert, H., Kohlmann, M., Wasternack, C. and Feussner, I.

(2000) Metabolic pro®ling of oxylipins upon sorbitol treatmentin barley leaves. Biochem. Soc. Trans. 28, 861±862.

Werck-Reichhart, D. and Feyereisen, R. (2000) Cytochromes P450:a success story. Genome Biol. 1, 3001.3±3001.9.

Winkler, R.G., Frank, M.R., Galbraith, D.W., Feyereisen, R. and

Feldmann, K.A. (1998) Systematic reverse genetics of transfer-DNA-tagged lines of Arabidopsis. Isolation of mutations in thecytochrome p450 gene superfamily. Plant Physiol. 118, 743±750.

Winz, R.A. and Baldwin, I.T. (2001) Molecular interactionsbetween the specialist herbivore Manduca sexta (Lepidoptera,Sphingidae) and its natural host Nicotiana attenuata. IV. Insect-induced ethylene reduces jasmonate-induced nicotineaccumulation by regulating putrescine N-methyltransferasetranscripts. Plant Physiol. 125, 2189±2202.

Zhao, D. and Ma, H. (2000) Male fertility: a case of enzymeidentity. Curr. Biol. 10, R904±R907.



A Knock-Out Mutation In Allene Oxide Synthase Results In Male Sterility and Defective Wound Signal...

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