The Plant Cell, Vol. 15, 1646–1661, July 2003, www.plantcell.org © 2003 American Society of Plant Biologists

The Tomato

Suppressor of prosystemin-mediated responses2

Gene Encodes a Fatty Acid Desaturase Required for the Biosynthesis of Jasmonic Acid and the Production of a Systemic Wound Signal for Defense Gene Expression

Chuanyou Li,

a

Guanghui Liu,

a

Changcheng Xu,

b

Gyu In Lee,

a

Petra Bauer,

c

Hong-Qing Ling,

d

Martin W. Ganal,

c,1

and Gregg A. Howe

a,b,2

a

Department of Energy Plant Research Laboratory, Michigan State University, East Lansing, Michigan 48824

b

Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824

c

Institute for Plant Genetics and Crop Plant Research, D-06466 Gatersleben, Germany

d

Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China

Genetic analysis of the wound response pathway in tomato indicates that systemin and its precursor protein, prosystemin, areupstream components of a defensive signaling cascade that involves the synthesis and subsequent action of the octadecatri-

enoic acid (18:3)–derived plant hormone jasmonic acid (JA). The

suppressor of prosystemin-mediated responses2

(

spr2

) muta-tion, which was isolated previously as a suppressor of (pro)systemin-mediated signaling, impairs wound-induced JA biosyn-

thesis and the production of a long-distance signal for the expression of defensive

Proteinase inhibitor

genes. Using amap-based cloning approach, we demonstrate here that

Spr2

encodes a chloroplast fatty acid desaturase involved in JAbiosynthesis. Loss of

Spr2

function reduced the 18:3 content of leaves to

�

10% of wild-type levels, abolished the accumu-lation of hexadecatrienoic acid, and caused a corresponding increase in the level of dienoic fatty acids. The effect of

spr2

on the fatty acyl content of various classes of glycerolipids indicated that the

Spr2

gene product catalyzes most, if not all,

�

3 fatty acid desaturation within the “prokaryotic pathway” for lipid synthesis in tomato leaves. Despite the reduced levelsof trienoic fatty acids,

spr2

plants exhibited normal growth, development, and reproduction. However, the mutant was com-promised in defense against attack by tobacco hornworm larvae. These results indicate that jasmonate synthesis fromchloroplast pools of 18:3 is required for wound- and systemin-induced defense responses and support a role for systeminin the production of a transmissible signal that is derived from the octadecanoid pathway.

INTRODUCTION

Many plants respond to insect attack and wounding by activat-ing the expression of genes involved in herbivore deterrence,wound healing, and other defense-related processes. The syn-thesis and deployment of wound-induced phytochemicals isregulated by signal transduction pathways that operate both lo-cally at the site of wounding and systemically in undamagedleaves (Green and Ryan, 1972). The occurrence of wound-induced defense responses in diverse plant species (Karbanand Baldwin, 1997) suggests the existence of common mecha-nisms to generate, transport, perceive, and transduce woundsignals into physiologically relevant changes in gene expres-sion. Wound-inducible defensive proteinase inhibitors (PIs) insolanaceous plants provide an attractive model system inwhich to study the mechanism of long-distance wound signal-ing and its relationship to plant defense (Ryan, 2000; Walling,

2000; León et al., 2001; Gatehouse, 2002; Kessler and Baldwin,2002; Li et al., 2002b).

A unique component of the wound response pathway inthese species is the peptide signal systemin and the precursorprotein prosystemin, from which it is derived (Pearce et al.,1991; McGurl et al., 1992). The tomato genome harbors a sin-gle copy of the

Prosystemin

(

Prosys

) gene that is transcribedand alternatively spliced to generate two functional forms of theprotein (Li and Howe, 2001). Several lines of genetic evidencedemonstrate that (pro)systemin performs an essential role inwound-induced defense responses. First, transgenic tomatoplants that express antisense

Prosys

cDNA are deficient inwound-induced systemic expression of PIs and exhibit in-creased susceptibility to herbivores (McGurl et al., 1992;Orozco-Cárdenas et al., 1993). Conversely, overexpression ofprosystemin from a

35S

:

Prosys

transgene constitutively acti-vates PI expression in unwounded plants and results in en-hanced resistance to herbivores (McGurl et al., 1994; Li et al.,2002a). Finally, forward genetic analysis in tomato has shownthat genes required for (pro)systemin-mediated signaling alsoare essential for wound-induced expression of

PI

and other de-fense-related genes (Howe et al., 1996; Howe and Ryan, 1999;Li et al., 2001; Lee and Howe, 2003).

1

Current address: TraitGenetics GmbH, Am Schwabeplan 1b, D-06466Gatersleben, Germany.

2

To whom correspondence should be addressed. E-mail howeg@msu.edu; fax 517-353-9168.Article, publication date, and citation information can be found atwww.plantcell.org/cgi/doi/10.1105/tpc.012237.

Role of

Spr2

in Systemin Signaling 1647

Transcriptional activation of

PI

genes in response to wound-ing and systemin depends on the action of jasmonic acid (JA)and related pentacyclic oxylipins (collectively referred to hereas jasmonates) that are derived from linolenic acid via the octa-decanoid pathway (Farmer and Ryan, 1992; Ryan, 2000; Li etal., 2001). The systemin/jasmonate signaling pathway is initi-ated upon the binding of systemin to a 160-kD plasma mem-brane–bound receptor (SR160) that was identified recently as amember of the Leu-rich repeat receptor–like kinase family ofproteins (Meindl et al., 1998; Scheer and Ryan, 1999, 2002).Binding of systemin to the cell surface is associated with sev-eral rapid signaling events, including increased cytosolic Ca

2

�

levels, membrane depolarization, inhibition of a plasma mem-brane proton ATPase, and activation of a mitogen-activatedprotein kinase activity (Felix and Boller, 1995; Moyen andJohannes, 1996; Stratmann and Ryan, 1997; Moyen et al.,1998; Schaller and Oecking, 1999).

How systemin triggers the release of linolenic acid frommembrane lipids for jasmonate biosynthesis remains unknown.The ability of systemin to activate phospholipase A

2

(PLA

2

) ac-tivity in tomato leaves suggests that systemin might effect therelease of linolenic acid from the plasma membrane, analogousto the PLA

2

-mediated release of arachidonic acid in animal sys-tems (Farmer and Ryan, 1992; Narváez-Vásquez et al., 1999).However, the demonstrated role of chloroplast-localized PLA

1

in JA biosynthesis during flower development in Arabidopsis(Ishiguro et al., 2001) raises the possibility that systemin per-ception at the plasma membrane is coupled to the activation ofa similar lipase in the chloroplast. In addition to systemin, oli-gogalacturonic acid (OGA) derived from plant cell walls acti-vates PI expression through the jasmonate signaling pathway(Bishop et al., 1981; Doares et al., 1995). JA synthesized in re-sponse to wounding, systemin, and OGA acts in concert withethylene (O’Donnell et al., 1996) and hydrogen peroxide(Orozco-Cárdenas et al., 2001) to positively regulate the ex-pression of downstream target genes.

The signal transduction pathway that regulates wound-induced PI expression in damaged tomato leaves is distinctfrom the pathway that operates in distal undamaged tissues (Liet al., 2002b; Strassner et al., 2002). Grafting experiments us-ing various wound-response mutants demonstrated that sys-temic PI expression requires jasmonate biosynthesis inwounded leaves but not in distal undamaged leaves (Li et al.,2002b). Conversely, the systemic response requires jasmonateperception in undamaged responding tissues but not in dam-aged leaves in which the long-distance signal is generated.These findings suggest that JA or a related compound derivedfrom the octadecanoid pathway functions as a non-cell-auton-omous wound signal. The wound-response phenotype of to-mato mutants that are defective in the production or perceptionof systemin indicates that this polypeptide functions primarily inthe systemic response (McGurl et al., 1992; Lee and Howe,2003). Homologs of tomato systemin have been identified inpotato, pepper, and nightshade but not in Arabidopsis or to-bacco (Constabel et al., 1998; Ryan et al., 2002). However, re-cent studies have demonstrated that tobacco contains twononhomologous peptide signals that are produced from a sin-gle precursor protein (Pearce et al., 2001). The systemin-like

bioactivity of these peptides suggests that they play a role inwound-induced systemic defense responses. In contrast tosystemins, OGAs are thought to mediate the JA-dependent ex-pression of

PI

genes in the vicinity of the wound site (Baydounand Fry, 1988; Ryan, 2000; Lee and Howe, 2003). Thus, OGAsand systemin appear to regulate the jasmonate pathway inways that promote the local and systemic expression, respec-tively, of wound-responsive defense genes.

Studies in Arabidopsis present a more complex view of woundsignaling than that proposed for solanaceous species. In particu-lar, wounding of Arabidopsis activates multiple signaling path-ways that regulate the expression of distinct sets of target genes.One pathway uses OGAs to induce the expression of wound-response genes at or near the site of wounding (Rojo et al., 1999).In contrast to tomato, in which JA and ethylene work synergisti-cally to coordinate wound-induced gene expression, OGA-induced signaling in Arabidopsis occurs independently of JA andethylene. Moreover, OGAs and ethylene appear to work togetherin Arabidopsis to negatively regulate JA-dependent wound re-sponses (Titarenko et al., 1997; Kubigsteltig et al., 1999; Rojo etal., 1999). Antagonism of the JA-dependent pathway by OGA andethylene in wounded leaves may provide a mechanism to regu-late the temporal and spatial expression of distinct sets ofwound-response genes (Rojo et al., 1999; León et al., 2001). Al-though jasmonates clearly are required for a subset of wound-induced systemic responses in both Arabidopsis and tomato,other remote responses to localized wounding in these plants arenot dependent on JA (Malone, 1996; Titarenko et al., 1997;O’Donnell et al., 1998; Howe et al., 2000; LeBrasseur et al., 2002).The physiological role of JA-independent wound responses, andtheir relationship to plant defense, remain to be determined.

The jasmonate pathway plays a central role in the regulationof induced defense responses in species throughout the plantkingdom (Gatehouse, 2002; Kessler and Baldwin, 2002; Liechtiand Farmer, 2002; Turner et al., 2002; Wasternack and Hause,2002). In this context, the apparent differences in the functionof ethylene, OGAs, and systemin in tomato versus Arabidopsismay reflect mechanisms that optimize the ability of each plantto control, through modulation of the jasmonate pathway, thetemporal and spatial expression patterns of target genes in re-sponse to a range of environmental stress conditions and de-velopmental cues. This idea is consistent with increasing evi-dence that jasmonates regulate different target processes indifferent plants. For example, genes that encode two of themajor classes of systemin- and JA-induced defense proteins intomato, ser proteinase inhibitor II (PI-II) and polyphenol oxi-dase, are absent in Arabidopsis (Allen, 2002; Van der Hoeven etal., 2002). It also has been noted that mutants of Arabidopsisthat are defective in the jasmonate pathway are male sterile,whereas comparable mutants in tomato are male fertile and, inone case, female sterile (Li et al., 2001; Wallis and Browse,2002). The role of systemin as a positive regulator of jasmonatesignaling in tomato offers a unique opportunity to understandpeptide-mediated responses in plants and to clarify the differ-ences between the wound signaling pathways in diverse plantspecies.

We are using a forward genetic approach to understand therole of systemin and JA in long-distance wound signaling and

1648 The Plant Cell

to determine how these signals interact to coordinate host de-fense responses. Toward this goal, we conducted a geneticscreen to identify mutations that suppress the constitutivewound-signaling phenotype of transgenic plants that overex-press a

35S

:

Prosys

transgene (Howe and Ryan, 1999). Amongseveral ethyl methanesulfonate–induced

spr

(

suppressor of pro-systemin-mediated responses

) mutants identified, one (

spr2

) wasshown to be impaired in wound-induced JA accumulation andthe production of the transmissible wound signal (Li et al.,2002b). Here, we show that

Spr2

encodes a chloroplast fattyacid desaturase that catalyzes the

�

3 desaturation of linoleicacid (18:2) to the jasmonate precursor, linolenic acid (18:3). Ourfindings demonstrate that wound- and systemin-induced acti-vation of anti-herbivore defense responses, as well as the pro-duction of the long-distance wound signal for

PI

expression,are dependent on chloroplastic pools of trienoic fatty acids thatgive rise to JA. The dependence of systemin action on

Spr2

isconsistent with the hypothesis that systemin functions to in-crease jasmonate synthesis to a level that is required for thesystemic response.

RESULTS

The Wound-Response Phenotype of

spr2

PlantsResults from a Defect in the Octadecanoid Pathwayfor JA Biosynthesis

The recessive

spr2

mutation was shown previously to sup-press the

35S

:

Prosys

-mediated expression of several defense-related genes, including the Ser

PI

genes

PI-I

and

PI-II

(Howeand Ryan, 1999). Further characterization of the mutant wasperformed using an

spr2

/

spr2

homozygous line in which the

35S

:

Prosys

transgene was removed by outcrossing, followedby three successive backcrosses to the wild-type cv Cas-tlemart. Aside from a slight chlorotic phenotype of youngleaves, the overall growth rate and morphology of

spr2

ho-mozygotes were indistinguishable from those of wild-typeplants. In response to mechanical wounding, however,

spr2

plants accumulated

�

5% of the wild-type levels of PI-II in thewounded leaf (local response) and no detectable PI-II in the up-per, unwounded leaf (systemic response; Figure 1A). Analysisof PI-II levels in an F2 population derived from a cross between

spr2

and the wild type showed that the ratio of wound-respon-sive wild-type to nonresponsive mutant plants was 89:34 (

�

2

�

0.46 for the 3:1 hypothesis). Consistent with the PI-II proteindata (Figure 1A), RNA gel blot analysis showed that

PI-II

wasexpressed very weakly in wounded

spr2

leaves and was notexpressed in systemic undamaged leaves (Figure 1B). The timecourse of this residual

PI

expression in the mutant was similarto that of wound-induced

PI-II

expression in wild-type plants,indicating that

spr2

affects the amplitude but not the timing ofwound-induced signaling. These results demonstrate that, inan otherwise wild-type genetic background,

spr2

behaves as asingle recessive mutation that impairs local and systemic

PI

gene expression in response to wounding.The deficiency in wound-induced JA accumulation in

spr2

plants (Li et al., 2002b) suggested that the mutation affects theoctadecanoid pathway for JA biosynthesis. To test this hypoth-

esis further, the responsiveness of

spr2

plants to various chem-ical elicitors of PI expression was determined. Systemin andOGA have been shown to activate PI expression via the octa-decanoid pathway (Doares et al., 1995). When supplied toyoung tomato plants through the cut stem, these compoundsinduced PI-II accumulation in wild-type but not

spr2

plants(Table 1). However,

spr2

plants were fully responsive to exoge-nous JA as well as its metabolic precursors 13(

S

)-hydroperoxylinolenic acid (13-HpOTrE) and 12-oxo-phytodienoic acid (12-OPDA). The mutant also expressed PI-II in response to be-statin, an amino peptidase inhibitor that activates

PI

gene ex-pression via a pathway that does not require JA production(Schaller et al., 1995). These results are fully consistent with thehypothesis that

spr2

abrogates wound-induced

PI

expressionby disrupting an early step in the octadecanoid pathway.

Recent studies have shown that tomato floral tissues consti-tutively accumulate high levels of JA, 12-OPDA, and jasmonateamino acid conjugates (Hause et al., 2000). To obtain additionalevidence for a role of

Spr2

in JA biosynthesis, gas chromatog-raphy–mass spectrometry was used to quantify the endoge-nous levels of JA in wild-type and

spr2

flowers. The level of JAin wild-type and

spr2

flowers of the same developmental stagewas 1485

�

290 and 265

�

36 pmol JA/g fresh weight of tis-sue, respectively. This result indicates that

spr2

disrupts notonly wound-induced JA synthesis in leaves but also constitu-tive JA production in reproductive tissues.

Map-Based Cloning of

Spr2

The deficiency in wound-induced PI-II protein accumulation in

spr2

plants provided a facile assay to identify the

Spr2

gene us-ing a positional cloning approach. A combination of amplifiedfragment length polymorphism and restriction fragment lengthpolymorphism (RFLP) markers was used to localize

Spr2

to aregion of

�

3 centimorgan between RFLP markers TG590 andTG118 (see Methods). A high-resolution map of the

Spr2

regionwas constructed by scoring a large mapping population (1436BC1 plants) for recombination events within the TG590-

Spr2

-TG118 interval (Figure 2). Markers showing cosegregation(258SL) or very tight linkage (TG590 and 149AR) to

Spr2

wereused to construct a BAC contig spanning the

Spr2

locus. Map-ping of sequences corresponding to the BAC ends showed that

Spr2

is located physically on BAC101L7. To identify putativegenes at the

Spr2

locus, the genomic insert of BAC101L7(

�

120 kb) was sequenced to approximately eightfold coverageusing a shotgun DNA-sequencing approach. A Basic LocalAlignment Search Tool (BLAST) search (Altschul et al., 1990) ofa 43-kb contiguous cosegregating genomic sequence againstthe tomato EST database (http://www.tigr.org) identified exactmatches to genes encoding phosphoribosylanthranilate isom-erase (EST275992), a putative nucleic acid binding protein(EST273694), and an

�

3 fatty acid desaturase (EST474001). Onthe basis of homology with the well-characterized

FAD7

genefrom Arabidopsis (Iba et al., 1993), the desaturase-encodinggene was designated

LeFad7

.

LeFad7

was considered to be a strong candidate for

Spr2

because (1)

�

3 desaturases convert 18:2 to the JA precursor,18:3, and (2)

spr2

plants are deficient in JA biosynthesis. Three

Role of

Spr2

in Systemin Signaling 1649

experiments were performed to test the hypothesis that

Spr2

isequivalent to

LeFad7

. First, an RFLP marker that distinguishes

LeFad7

in

Lycopersicon pennellii

(

Fad7

pen

) from the tomato al-lele (

Fad7

esc

) was mapped using the recombination eventswithin the TG590-TG118 interval. The results showed that thegenotype of all wound-responsive BC1 plants was

Fad7

pen

/

Fad7

esc

, whereas the genotype of all mutant plants was

Fad7esc/Fad7esc. This finding suggested that LeFad7 and Spr2are genetically identical. Second, comparison of the sequenceof a LeFad7 cDNA obtained from spr2 plants with the wild-typecDNA revealed the existence of a single nucleotide differencebetween the two sequences. This polymorphism, which wasconfirmed in the genomic exon sequence from wild-type andspr2 plants, resulted in the transition of a G to an A at nucle-otide 12 of the open reading frame. This mutation changed thecodon TGG, which specifies Trp, to the stop codon TGA, re-sulting in premature termination of the protein. This mutation is

predicted to abolish the function of LeFad7 and thus could ac-count for the JA deficiency in spr2 plants.

To confirm this possibility, experiments were performed to de-termine whether the wild-type LeFad7 gene would complementthe spr2 mutant phenotype. For this purpose, a 2.05-kb genomicfragment containing the wild-type LeFad7 gene was placed un-der the control of the 35S promoter of Cauliflower mosaic virus inthe T-DNA vector pBI121. This construct was introduced into thespr2 genetic background using Agrobacterium tumefaciens–mediated transformation. DNA and RNA gel blot analyses identi-fied six independent kanamycin-resistant transformants (T0) thatexpressed one or more copies of the 35S:LeFad7 transgene(data not shown). All six of these lines showed normal levels oflocal and systemic PI-II accumulation in response to mechanicalwounding (Figure 3). This result demonstrates that 35S:LeFad7complements the wound-signaling defect in spr2 plants, provingthat Spr2 and LeFad7 are equivalent.

Figure 1. spr2 Impairs the Wound-Induced Expression of Proteinase Inhibitors.

(A) PI-II accumulation in tomato leaves in response to mechanical wounding. Sixteen-day-old wild-type (WT) and spr2 plants were wounded with ahemostat. Twenty-four hours after wounding, PI-II levels were measured in the wounded leaf (light gray bars; L, local response) and an upper, un-wounded leaf (dark gray bar; S, systemic response). As controls (C), PI-II levels were measured in leaf juice expressed from pooled upper and lowerleaves of unwounded plants (black bar). Values represent means � SD from 12 plants per genotype.(B) Time course of PI-II transcript levels in response to mechanical wounding. Sixteen-day-old seedlings of wild-type (WT) and spr2 plants were me-chanically wounded at the distal end of the terminal leaflet of the lower leaf. At various times thereafter, the lower damaged leaf (L, local response) andan upper, unwounded leaf (S, systemic response) were harvested separately for RNA extraction. RNA gel blots were hybridized to a 32P-labeled PI-IIcDNA or, as a loading control, a cDNA for translation initiation factor eIF4A.

1650 The Plant Cell

Spr2/LeFad7 Encodes a Chloroplast �3 FattyAcid Desaturase

�3 fatty acid desaturases catalyze the conversion of dienoic fattyacids to trienoic fatty acids both in the chloroplast via the so-called prokaryotic pathway and in the endoplasmic reticulum (ER)via the eukaryotic pathway (Browse and Somerville, 1991; Wallisand Browse, 2002). Although plastid- and ER-localized �3 desatu-

rases share significant sequence similarity, they form distinct phy-logenetic groups (Figure 4A). The Spr2/LeFad7 gene (henceforthreferred to as LeFad7) is predicted to encode a 435–amino acidprotein (LeFAD7) that clearly belongs to the chloroplast group ofdesaturases. The deduced amino acid sequence of LeFAD7 ismost similar to those of chloroplast �3 desaturases from othersolanaceous plants, including potato (94% identity; Martín et al.,1999), tobacco (82% identity; Hamada et al., 1996), and pepper(81% identity; Kwon et al., 2000). Similar to other chloroplast �3desaturases, the N terminus of LeFAD7 has characteristic featuresof a chloroplast-targeting sequence (Keegstra et al., 1989). Aplastidic location for the protein was further supported by com-puter analyses using the ChloroP (http://www.cbs.dtu.dk/services/ChloroP/) and TargetP (http://www.cbs.dtu.dk/services/TargetP/)programs.

Comparison of the genomic and cDNA sequences revealedthat the LeFad7 gene contains seven exons and six introns(Figure 4B). The coding capacity of individual exons, as well asthe positions of intron sequences within the gene, indicatedthat LeFad7 is homologous with the well-characterized FAD7and FAD8 genes that encode chloroplast-localized �3 desatu-rase isozymes in Arabidopsis (Figure 4B) (Iba et al., 1993; Gibsonet al., 1994). A notable difference between the tomato and Ara-bidopsis genes was the presence of an additional intron at the5� end of AtFAD7 and AtFAD8. Genomic DNA hybridization ex-periments using the LeFad7 cDNA as a probe suggested thatthis gene exists as a single copy in the tomato genome (data

Table 1. Proteinase Inhibitor II Accumulation in Wild-type and spr2 Plants in Response to Elicitors

Elicitor Wild Type spr2

Buffer 16 � 10 0Systemin 135 � 26 0OGA 92 � 15 013-HpOTrE 79 � 10 71 � 912-OPDA 126 � 18 119 � 21JA 142 � 24 136 � 19Bestatin 115 � 9 100 � 16

Fifteen-day-old seedlings were supplied with buffer (15 mM sodiumphosphate, pH 6.5), systemin (5 pmol/plant), OGA (20 g/plant), 13-HpOTrE (25 nmol/plant), 12-OPDA (10 nmol/plant), JA (10 nmol/plant),or bestatin (60 nmol/plant) through the cut stem. PI-II levels (g/mL leafjuice) were measured in leaf juice expressed from leaves 24 h later. Val-ues indicate means � SD of at least 12 plants. The detection limit of thePI-II assay was �5 g of PI-II/mL of leaf juice.

Figure 2. Map-Based Cloning of the Spr2 Gene.

The positions of molecular markers within the TG590-TG118 interval relative to the position of Spr2 on chromosome 6 are shown at top. Numbers be-low the line indicate the number of recombination events identified among 1436 BC1 plants analyzed. The positions of markers corresponding to theright (R) and left (L) ends of yeast artificial chromosome (YAC) and BAC clones were determined by mapping experiments using the 39 recombinationevents in the TG590-TG118 interval. The dotted vertical line at bottom denotes a BAC end that could not be mapped as a result of repetitive se-quences. Placement of Spr2 on BAC101L7 was determined by the phenotypic data associated with recombination events 567 and 643.

Role of Spr2 in Systemin Signaling 1651

not shown). Consistent with this notion, all 42 tomato ESTs an-notated as chloroplast �3 fatty acid desaturases constitute asingle tentative consensus sequence (TC99514; release of April25, 2002) that exactly matches the sequence of LeFad7. A sur-vey of the tomato EST database also revealed the existence ofanother gene (TC99736) that encodes a putative ER-localized�3 fatty acid desaturase, designated LeFAD3 (Figure 4A).

LeFad7 Is Expressed Constitutively in Various Tissues

RNA gel blot analysis was used to determine the expression pat-tern of LeFad7 in various tissues of tomato. In wild-type plants,LeFad7 transcripts were relatively abundant in leaf, petiole, andflower, with somewhat lower expression observed in stems androots (Figure 5A). This broad expression pattern is consistent withthe fact that the ESTs corresponding to LeFad7 were identified incDNA libraries constructed from leaf, fruit, flower, ovary, pollen,trichome, germinating seed, and root (http://www.tigr.org/). Thelevel of LeFad7 mRNA accumulation in tissues from spr2 plantswas significantly less than that in the corresponding wild-type tis-sues (Figure 5A). This observation is consistent with the well-doc-umented existence of RNA surveillance mechanisms that targetmRNAs containing premature termination codons for rapid deg-radation (Hilleren and Parker, 1999).

In leaf tissue of several plants, transcripts that encode chlo-roplast �3-desaturases have been shown to accumulate in re-sponse to wounding (Hamada et al., 1996; Nishiuchi et al.,1997; Martín et al., 1999; Kwon et al., 2000). To determinewhether LeFad7 expression is wound inducible, we mechani-cally wounded wild-type plants and measured the levels ofLeFad7 transcript by RNA gel blot analysis at various timesthereafter (Figure 5B). This experiment showed that wounding

does not significantly affect the steady state level of LeFad7mRNA in tomato leaves. Hybridization of the same RNA gelblots to a probe for PI-II confirmed that the treatment was ef-fective in activating wound-induced gene expression.

spr2 Impairs the Desaturation of Dienoic Fatty Acidsin Chloroplast Lipids

To determine the effect of spr2 on the biosynthesis of polyun-saturated fatty acids, the levels of various C16 and C18 fattyacids in total lipids of wild-type and spr2 leaves were assessedby gas chromatography (Figure 6A). The major fatty acid com-ponents of the total lipids in wild-type tomato leaves were 18:3,18:2, hexadecatrienoic acid (16:3), and palmitic acid (16:0),consistent with previous studies on tomato (Whitaker, 1986;Conconi et al., 1996; Ouariti et al., 1997). In spr2 leaves, 18:3constituted �5% of the total fatty acid content, whereas theaccumulation of 16:3 was undetectable (Figure 6A). The de-creased content of trienoic fatty acids in spr2 leaves was ac-companied by a corresponding increase in the level of dienoicfatty acids (i.e., 16:2 and 18:2). With the exception of a smallbut significant increase in the proportion of oleic acid (18:1) inspr2 leaves, the levels of other C16 and C18 fatty acids in themutant were comparable to wild-type levels. To determinewhether the spr2 mutation was responsible for the altered fattyacid composition of spr2 leaves, the total fatty acyl content inleaves of spr2 transgenic lines expressing the 35S:LeFad7transgene was determined (Figure 6A). The results showed that35S:LeFad7 expression in the spr2 genetic background re-stored the normal profile of fatty acids in total leaf lipids in eachof the transgenic lines tested.

To assess the contribution of LeFAD7 to fatty acid desatura-tion in nonphotosynthetic tissues, we compared the fatty acylcontent of total lipids from wild-type and spr2 roots. The majorlipid-derived fatty acids in wild-type roots were 18:2 (48.4%),16:0 (26.1%), and 18:3 (15.0%) (Figure 6B). This profile is es-sentially identical to that reported by Ouariti et al. (1997). Thefatty acid composition of spr2 roots was very similar to that ofwild-type roots, with only minor differences (�5% deviationfrom wild-type composition) observed in the relative propor-tions of 18:2 and 18:3. These results indicate that LeFAD7functions primarily in the desaturation of lipids in leaves andother photosynthetic tissues.

To gain additional insight into the biochemical function ofLeFAD7 in lipid biosynthesis, we separated individual glycerolip-ids from wild-type and spr2 leaves and analyzed them for fattyacid composition (Table 2). The results showed that all major lip-ids synthesized in spr2 leaves contained reduced levels of 18:3and a corresponding increase in the proportion of 18:2. It wasapparent, however, that spr2 had a greater effect on the desatu-ration of lipids that are synthesized in the chloroplast (monoga-lactosyldiacylglycerol [MGDG], digalactosyldiacylglycerol [DGDG],sulfoquinovosyldiacylglycerol [SQDG], and phosphatidylglycerol[PG]) than it does on lipids such as phosphatidylcholine (PC),phosphatidylinositol (PI), and phosphatidylethanolamine (PE)that accumulate primarily in extrachloroplastic membranes. Forexample, the proportion of 18:3 in chloroplast galactolipids(MGDG and DGDG) from the mutant ranged from 5 to 8% of that

Figure 3. Functional Complementation of spr2 with 35S:LeFad7.

Wound-induced PI-II accumulation in leaves from wild-type (WT), spr2,and 35S:LeFad7-expressing spr2 plants. Plants of each genotype (atthe five- to six-leaf stage) were wounded once across the midvein ofeach leaflet on the two lower leaves. Wounding of the same leaflets wasrepeated 3 h later. One day after the wound treatment, PI-II levels weredetermined both in the wounded leaflets (black bars) and in leaflets fromthe upper, unwounded leaves (white bars). Values represent means �SD from six independent transgenic T0 lines. PI-II levels in unwoundedcontrol plants were below the detection limit of the assay (�5 g of PI-II/mL of leaf juice).

1652 The Plant Cell

found in the corresponding lipids from wild-type plants, whereasthe proportion of 18:3 in extrachloroplast lipids (PC, PI, and PE)ranged from 28 to 55% of wild-type levels (Table 2). These anal-yses also showed that spr2 abolished the accumulation of 16:3,which in wild-type plants is found predominantly in the sn-2 po-sition of the chloroplast-specific lipid MGDG (Jamieson andReid, 1971; Browse and Somerville, 1991). These results providedirect biochemical evidence for a role of LeFAD7 in the desatura-tion of dienoic fatty acids in the chloroplast. The spr2 mutationappeared to have little or no effect on the level of stearic acid(16:0 and 18:0) in any of the lipid classes examined. Consistentwith the analysis of total fatty acids (Figure 6A), however, the

amount of 18:1 in some lipids (e.g., MGDG) was increasedslightly in spr2 leaves relative to wild-type levels.

Loss of LeFad7 Function Results in Increased Susceptibilityto Insect Attack

The inability of spr2 plants to express significant levels of de-fensive PIs in response to mechanical wounding (Figure 1) sug-gested that the mutant might be compromised in resistance toherbivorous insects. To test this possibility, 3-week-old (experi-ment 1) and 6-week-old (experiment 2) wild-type and spr2plants were challenged with tobacco hornworm larvae. After

Figure 4. Spr2 Encodes a Chloroplast �3 Fatty Acid Desaturase.

(A) Phylogenetic relationship of the Spr2 gene product, LeFAD7, to other plant �3 fatty acid desaturases. Shown is an unrooted neighbor-joining phy-logenic tree constructed in PAUP4.0* from the predicted amino acid sequences of representative desaturases. The phylogenic groups on the left andright sides of the tree correspond to ER-localized and chloroplast-localized �3 desaturases, respectively. Sequences were from Arabidopsis (AtFAD3,AtFAD7, and AtFAD8), potato (StFAD7), rice (OsFAD3), wheat (TaFAD3 and TaFAD7), maize (ZmFAD7 and ZmFAD8), perilla (PfFAD7), soybean(GmFAD3 and GmFAD7), tobacco (NtFAD3 and NtFAD7), sesame (SiFAD7), and tomato (LeFAD7 and putative LeFAD3).(B) Gene structure of Spr2/LeFad7 compared with homologous FAD7 and FAD8 genes from Arabidopsis. Intron and exon sequences are indicated byhorizontal lines and closed boxes, respectively, and are drawn to scale. Numbers above and below each gene denote the number of nucleotides ineach exon and intron, respectively. Only the translated portion of the first and last exon of each gene is shown, together with the start (ATG) and stop(TAG or TGA) codons within the respective exons.

Role of Spr2 in Systemin Signaling 1653

termination of the feeding trial, PI-II protein accumulation indamaged leaf tissue was measured, as was the weight gain oflarvae reared on the two host genotypes. In contrast to highlevels of PI-II accumulation in herbivore-damaged wild-typeleaves, no PI-II accumulation was detected in leaves from dam-aged spr2 plants (Table 3). The average weight of larvae rearedon spr2 plants was 2.3-fold (experiment 1) and 3.1-fold (experi-ment 2) greater than that of larvae reared on wild-type plants.Furthermore, it was apparent that larvae consumed more foli-age from the mutant plants than from the wild-type plants (Fig-ure 7). These data indicate that spr2 compromises the plant’sdefense response against herbivory. As a result, foliage fromspr2 plants is a better food source for hornworm larvae.

DISCUSSION

Spr2 Encodes a Chloroplast �3 Fatty Acid Desaturase

In the present study, we used a positional cloning approach todemonstrate that the Spr2 gene of tomato encodes a fatty aciddesaturase (designated LeFAD7) that is required for jasmonatebiosynthesis. Several observations lead us to conclude thatLeFAD7 functions within the chloroplast to catalyze the conver-sion of 16:2 and 18:2 to 16:3 and 18:3, respectively. First, thededuced amino acid sequence of LeFAD7 showed a high de-gree of similarity to several well-characterized chloroplast �3

desaturases and was phylogenetically distinct from the groupof ER-localized �3 desaturases. The structure of the LeFad7gene also indicated that it is homologous with the FAD7 andFAD8 genes of Arabidopsis that encode chloroplast �3 desatu-rase isozymes. Second, the spr2 mutation, which abolishes thefunction of LeFAD7, causes a massive decrease in 16:3 and18:3 and a corresponding increase in the levels of 16:2 and18:2 in leaf lipids. Particularly noteworthy was the absence inspr2 leaves of detectable levels of 16:3, which is synthesizedexclusively in the chloroplast (Browse and Somerville, 1991).Third, the 18:2 and 18.3 content of spr2 roots differed onlyslightly from that of wild-type roots (Figure 6B). Because plastidsare a minor component of the fatty acid content in roots, thisobservation supports the idea that LeFAD7 is a chloroplast de-saturase. Finally, a role for LeFAD7 in the synthesis of trienoicfatty acids in the chloroplast is consistent with the fact that theoctadecanoid pathway for JA biosynthesis is initiated in thiscellular compartment (Weber, 2002).

Much of what is known about trienoic fatty acid biosynthesisin plants has come from the characterization of Arabidopsis mu-tants that are deficient in one or more of three �3 desaturases(FAD3, FAD7, and FAD8) encoded by the Arabidopsis genome(Wallis and Browse, 2002). The FAD3 gene product is an ER-localized enzyme that desaturates 18:2, whereas the FAD7 geneproduct desaturates both 16:2 and 18:2 in the chloroplast.FAD8 is an isozyme of FAD7 that is expressed predominantly at

Figure 5. LeFad7 Is Expressed Constitutively in Various Tissues.

(A) Blots containing total RNA from root, stem, petiole, and leaf tissue from 16-day-old wild-type (WT) and spr2 plants were probed with the LeFad7cDNA. Flower RNA was obtained from mature unopened or newly opened flowers harvested from 7- to 8-week-old plants. Blots also were hybridizedto a probe for eIF4A as a loading control.(B) Expression of LeFad7 and PI-II in response to wounding. Sixteen-day-old wild-type seedlings were wounded on both lower and upper leaves, and to-tal RNA was extracted from the wounded tissue at the times indicated (h) thereafter. RNA was subjected to RNA gel blot analysis as described for (A).

1654 The Plant Cell

low temperature (McConn et al., 1994). Our results strongly sug-gest that LeFAD7 is functionally homologous with the Arabidop-sis FAD7 gene product. In this context, it is of interest to com-pare tomato and Arabidopsis with respect to the effects thatmutations in these genes have on the trienoic fatty acid contentof leaves. Both species are considered 16:3 plants owing to therelatively high abundance of the 16:3 acyl group in chloroplast-

specific galactolipids (Jamieson and Reid, 1971). At standardlaboratory temperatures, the fatty acyl content of leaves fromArabidopsis fad7 plants contains �2% 16:3 and 19% 18:3,whereas fad7 fad8 double mutants contain no 16:3 and �17%18:3 (McConn et al., 1994). By comparison, leaves of the tomatospr2 mutant contain no detectable 16:3 and only �5% 18:3.This finding indicates that the contribution of LeFAD7 to the pro-duction of trienoic fatty acids in tomato leaves is greater thanthe combined contribution of FAD7 and FAD8 in Arabidopsisleaves. Thus, unlike in Arabidopsis, there appears to be rela-tively little genetic or biochemical redundancy in the pathway fortrienoic fatty acid synthesis in tomato leaves.

Plant cells use both a chloroplast-localized prokaryotic path-way and an ER-localized eukaryotic pathway for the synthesisof glycerolipids and the associated production of trienoic fattyacids. Metabolic coordination between these pathways isachieved, at least in part, by lipid transport between chloroplastand extrachloroplast membranes (Roughan et al., 1980; Browseand Somerville, 1991). The effect of spr2 on the fatty acyl con-tent of various lipids that constitute chloroplast and extrachlo-roplast membranes is consistent with this idea. For example,transport of a portion of the 18:3-containing lipids synthesizedin the ER to the chloroplast may account for the residual levelof 18:3 in chloroplast-specific lipids of spr2 leaves. Such amechanism was proposed previously to account for the 18:3content of chloroplast lipids in Arabidopsis mutants that are im-paired in chloroplast �3 desaturase activity (Somerville andBrowse, 1991; McConn et al., 1994). By comparison with Ara-bidopsis, the relatively low level of 18:3 in chloroplast lipidsfrom spr2 leaves suggests that the contribution of the eukary-otic pathway to chloroplast lipid synthesis in tomato is lessthan that in Arabidopsis. It also is possible that tomato containsa second chloroplast �3 desaturase that is analogous to FAD8

Figure 6. Fatty Acid Composition in Lipids from Wild-Type and spr2Plants.

(A) Lipids were extracted from young leaves of 4-week-old wild-type(black bars), spr2 (light gray bars), and 35S:LeFad7-expressing spr2 (darkgray bars) plants. Fatty acids obtained by lipid saponification were quanti-fied by gas chromatography as their methyl ester derivatives. The y axisshows the mole percent of each fatty acid species indicated on the ab-scissa. Values represent means � SD from three plants per genotype.(B) Lipids were extracted from roots of 18-day-old wild-type (blackbars) and spr2 (white bars) plants and analyzed as described for (A).Values represent means � SD from six plants per genotype.

Table 2. Fatty Acid Composition of Lipids from Wild-Type andspr2 Leaves

Fatty acid compositiona

Lipid Genotype 16:0 16:1 16:2 16:3 18:0 18:1 18:2 18:3

MGDG Wild type 6.7 1.2 –b 15.8 0.8 0.7 3.7 71.1spr2 6.0 2.1 0.3 – 1.1 3.2 83.4 3.8

DGDG Wild type 31.2 0.8 0.2 1.0 1.5 1.6 3.4 60.1spr2 26.5 1.0 0.1 – 1.4 2.4 63.9 4.6

SQDG Wild type 70.7 – 0.4 2.3 0.5 4.2 7.9 14.1spr2 63.7 0.8 0.3 – 1.3 3.9 28.4 1.4

PG Wild type 26.7 32.4 – 1.0 – 4.8 21.1 14.0spr2 25.0 30.0 0.3 – 0.9 8.6 33.7 0.8

PC Wild type 35.7 0.8 0.2 0.6 1.3 7.0 40.9 13.6spr2 33.7 0.8 0.2 – 1.3 6.9 53.3 3.8

PI Wild type 58.2 0.4 1.4 2.0 3.0 8.7 21.9 4.5spr2 57.2 – 0.7 – 2.5 6.7 30.4 2.5

PE Wild type 42.5 0.7 0.6 1.0 1.0 3.6 41.7 8.9spr2 42.4 0.7 0.3 – 1.1 3.7 48.0 3.7

a Values represent means of triplicate samples and are presented asmole percent. Standard deviations between triplicates was �2% of theindicated values.b Present at trace levels (�0.1% of total fatty acid).

Role of Spr2 in Systemin Signaling 1655

of Arabidopsis. However, genomic DNA hybridization experi-ments and surveys of the tomato EST database failed to pro-vide evidence for the existence of such a gene. The inability todetect 16:3 in lipids from spr2 leaves also contradicts this pos-sibility. Another implication of the results presented in Table 2is that LeFAD7 produces trienoic fatty acids for both chloro-plast and extrachloroplast membranes. The reduced 18:3 con-tent of extrachloroplast lipids (i.e., PI, PE, and PC) in spr2leaves supports previous work in Arabidopsis (Browse et al.,1986), suggesting that there is substantial flux of 18:3 betweenthe chloroplast and extrachloroplast membranes.

Role of Trienoic Fatty Acids in Systemin- andWound-Induced Signaling

The results presented here confirm the previous proposal thatspr2 abrogates wound- and systemin-induced gene expressionby blocking JA biosynthesis (Li et al., 2002b). This conclusion issupported by the deficiency in JA production in spr2 leaves andflowers as well as by the ability of the mutant to respond to ex-ogenous JA. Our conclusion that LeFad7 function is essential forwound-induced PI expression is consistent with results obtainedusing potato transgenic lines in which the expression of a chloro-plast �3 desaturase was reduced by antisense technology(Martín et al., 1999). In the case of tomato, it is noteworthy thatthe small amount of 18:3 in spr2 chloroplast lipids (�7% of thecombined 18:3 levels in MGDG, DGDG, sulfolipid [SL], and PG inwild-type leaves) was correlated with the capacity of spr2 leavesto accumulate low levels (�7% of wild-type levels) of JA in re-sponse to wounding (Li et al., 2002b). This residual amount of JAlikely accounts for the low level of PI expression in wounded spr2leaves. Because LeFad7 appears to be a single-copy gene, a pu-tative function for the ER-resident �3 FAD3 in generating 18:3that is used for the production of low levels of JA in leaves, aswell as the synthesis of 18:3 in roots, can be proposed.

The broad effect of spr2 on the trienoic fatty acid content of lip-ids desaturated in the chloroplast (i.e., MGDG, DGDG, SL, and

Table 3. Tobacco Hornworm Feeding Assay on Wild-type andspr2 Plants

Experimenta Genotype PI-II (g/mL) Larval Weight (g)b

1 Wild type 157 � 15 0.53 � 0.19 (n � 25)spr2 0 1.21 � 0.25 (n � 21)

2 Wild type 303 � 25 1.82 � 0.27 (n � 14)spr2 0 5.58 � 0.67 (n � 16)

a In experiment 1, 30 newly hatched larvae were placed randomly on theleaves of 30 3-week-old tomato plants of each genotype. Larvae wereallowed to move freely between plants of the same genotype. In experi-ment 2, six newly hatched larvae were placed on leaves of each of fourseparately potted 6-week-old plants of each genotype. Experiments 1and 2 were terminated 10 and 12 days after the start of the feeding trial,respectively. At that time, PI-II levels in damaged leaf tissue were mea-sured, as was the weight of larvae recovered from the plants. Data rep-resent means � SD. The detection limit of the PI-II assay was �5 g PI-II/mL leaf juice.b In both experiments, the weight of larvae grown on wild-type and mu-tant plants was significantly different at P � 0.001 (Student’s t test).

PG) indicates that LeFAD7, like the FAD7 enzyme in Arabidopsis(Browse et al., 1986), is not specific for the nature of the lipid headgroup or the length of the fatty acid. Given this feature of the en-zyme, our results do not provide insight into the identity of theparticular class of chloroplast lipid that provides the 18:3 sub-strate for JA synthesis. However, constitutive expression ofLeFad7 transcript in leaf tissue supports the idea that wound-and systemin-mediated defense gene expression does not re-quire the de novo synthesis of 18:3 but rather involves new syn-thesis of JA from existing pools of lipid-esterified 18:3 or an18:3-derived intermediate in the octadecanoid pathway. Thesedata are in general agreement with previous work showing thatwound-induced JA accumulation in tomato leaves is correlatedwith the release of 18:3 and 18:2, most likely from chloroplast lip-ids (Conconi et al., 1996). The preponderance of 18:2 in chloro-plast lipids from spr2 leaves indicates that oxylipins derived fromthis polyunsaturate (Blechert et al., 1995) are not active as signalsfor PI gene expression in response to wounding and systemin.

The spr2 mutation was identified initially on the basis of itsability to suppress the systemin-mediated expression of defen-sive genes (Howe and Ryan, 1999). Identification of the Spr2gene product as a chloroplast �3 desaturase provides strong ev-idence that systemin signaling involves the synthesis of JA froma pool of 18:3 located in chloroplast lipids rather than extrachlo-roplast lipids. This interpretation is supported by the observationthat spr2 affects the 18:3 content of chloroplast lipids muchmore than that of extrachloroplast lipids. The precise mechanismby which systemin rapidly induces the massive accumulation ofJA (Doares et al., 1995) remains to be determined. However, theinvolvement of the chloroplast in systemin-mediated defense re-sponses implies the existence of a signaling cascade that cou-ples the activation of the systemin receptor (i.e., SR160) at theplasma membrane to the initiation of JA biosynthesis in theplastid. Wound-inducible mitogen-activated protein kinase andphospholipase activities have been implicated in this aspectof systemin signaling (Stratmann and Ryan, 1997; Narváez-Vásquez et al., 1999). Further characterization of mutants thatare impaired in systemin perception (Lee and Howe, 2003) mayprovide additional insight into the signaling pathway that linkssystemin action to the activation of the jasmonate pathway.

Molecular cloning of Spr2 provides new insight into the role ofJA biosynthesis in wound-induced expression of systemic de-fense responses. Of relevance to this issue are grafting studiesshowing that spr2 impairs production of the transmissible woundsignal for PI expression (Li et al., 2002b). Identification of Spr2 asa chloroplast �3 desaturase provides solid support for the hy-pothesis that jasmonate synthesis is required for the productionof the systemic wound signal in wounded leaves. It also is note-worthy that spr2 does not affect the ability of grafted scions toperceive the long-distance signal that emanates from woundedleaves (Li et al., 2002b). Because spr2 impairs JA synthesis byblocking 18:3 production, it can be inferred that de novo synthe-sis of JA (or of related compounds derived from trienoic fatty ac-ids) is not required in cells that perceive and respond to the sys-temic signal. This interpretation is in agreement with the work ofStrassner et al. (2002), who used gene expression profiling anddirect measurements of jasmonates to study wound-inducedgene expression in tomato plants. The capacity of spr2 plants to

1656 The Plant Cell

perceive the systemic wound signal, together with the inabilityof the mutant to respond to exogenous systemin or 35S:Prosys(Howe and Ryan, 1999), strongly suggests that jasmonate ratherthan systemin is the transmissible wound signal for PI gene ex-pression. Recent studies of the systemin-insensitive mutant spr1indicate that systemin may function at or near the site of wound-ing (i.e., in rootstock tissues) to activate JA synthesis to a levelthat is required for the systemic response (Lee and Howe, 2003).It is possible that systemin and jasmonate are both mobile com-ponents of the signaling pathway and that positive feedback in-teractions between them serve to propagate the long-distancesignal (Ryan and Moura, 2002).

Because spr2 impairs the production of trienoic fatty acids, wecannot exclude the possibility that some defense-related pheno-types of the mutant are attributed to a deficiency in 16:3/18:3-derived oxylipins other than JA. For example, the cyclopenten-one 12-OPDA has been shown to exhibit biological activity in theabsence of prior conversion to JA (Blechert et al., 1995, 1999;Stintzi et al., 2001). Increasing evidence also indicates that C6 al-dehydes produced from the hydroperoxide lyase branch of oxy-lipin metabolism play an important role in plant defense signaling(Bate and Rothstein, 1998; Vancanneyt et al., 2001). Exogenoustrans-2-hexenal, a C6 oxylipin produced in tomato by the se-quential action of 13-lipoxygenase and hydroperoxide lyase, wasshown recently to trigger local and systemic emission of mono-terpenes and sesquiterpenes that are implicated in the attractionof natural predators of herbivores (Farag and Paré, 2002). Thespr2 mutant should provide a valuable tool with which to assessthe function of the complete repertoire of oxylipins that are pro-duced endogenously from trienoic fatty acids. Based on thetrienoic fatty acid deficiency in spr2 leaves, it also can be pre-dicted that the mutant is impaired in the synthesis of 16:3-derived oxylipins such as dinor-OPDA (Weber et al., 1997). Nev-ertheless, the ability of spr2 plants to respond to exogenous JAand its C18 precursors (Table 1) is consistent with previous stud-ies indicating that JA is the active signal for the expression of PIgenes in tomato leaves (Farmer and Ryan, 1992; Conconi et al.,1996; Wasternack et al., 1998; Strassner et al., 2002).

Role of Trienoic Fatty Acids in the Growthand Development of Tomato

The spr2 mutant provides a useful resource to investigate thebroader role of trienoic fatty acids in plant growth and develop-ment. The rationale underlying this approach has been illus-trated by the elegant use of comparable Arabidopsis mutantsto study the role of polyunsaturates in temperature toleranceand photosynthesis (Browse and Somerville, 1991; Wallis andBrowse, 2002). The slightly chlorotic phenotype of young spr2leaves is similar to that of �3 desaturase mutants of Arabidop-sis (Wallis and Browse, 2002) and clearly suggests a role forLeFAD7 in chloroplast function. With regard to temperature tol-erance, it is interesting that tomato suffers from chilling injurywhen exposed to temperatures of �10C or less and has littlecapacity to adapt to cold temperatures (Jaglo et al., 2001). Inmany plants, including Arabidopsis, the capacity to synthesizepolyunsaturated fatty acids is an important component of low-temperature fitness (Hugly and Somerville, 1992; Miquel et al.,

Figure 7. Challenge of Wild-Type and spr2 Plants with Tobacco Horn-worm Larvae.

Six newly hatched hornworm larvae (�5 mg each) were placed on eachof four plants (6 weeks old) of each genotype. Larvae were allowed tofeed for 12 days and remained on the same plant for the duration of thetrial as described for experiment 2 in Table 3. WT, wild type.(A) Representative wild-type and spr2 plants at the end of the feeding trial.(B) Hornworm larvae recovered at the end of the trial from representa-tive wild-type and spr2 plants.

Role of Spr2 in Systemin Signaling 1657

1993; Routaboul et al., 2000). It has been proposed that thecold-inducible FAD8 gene of Arabidopsis may provide in-creased �3 desaturase activity for optimal growth at low tem-peratures (Gibson et al., 1994). The apparent absence of aFAD8 homolog in tomato, which is of tropical origin, may con-tribute to the cold-sensitive trait and the inability of the plant toaccumulate increased levels of trienoic fatty acids in responseto low temperatures (Novitskaya et al., 2000).

The spr2 mutant also may provide insight into the role of JAand related oxylipins in development. It will be interesting todetermine whether the various developmental roles fulfilled byjasmonates in Arabidopsis also apply to tomato, a solanaceousspecies that diverged from the lineage leading to Arabidopsisas many as 150 million years ago (Yang et al., 1999). It is wellestablished that the biosynthesis and perception of JA is es-sential for male reproductive development in Arabidopsis(McConn and Browse, 1996; Wallis and Browse, 2002). Despitethe 18:3 deficiency in spr2 plants, however, this mutant dis-played normal fertility, as determined by seed set of self-polli-nated spr2 plants and by outcrossing of spr2 pollen to a wild-type female parent. It is possible that the residual amount of JA(18% of wild-type levels) in spr2 flowers is sufficient to promotefertility. However, this interpretation is not consistent with theobservation that the PLA1-deficient dad1 mutant of Arabidopsisexhibits �22% of wild-type levels of JA in flowers but is malesterile (Ishiguro et al., 2001). It also is possible that male fertilityin tomato involves JA derivatives such as JA amino acid conju-gates or 12-OPDA that accumulate to high levels in tomatoflowers (Hause et al., 2000). Measurement of the endogenouslevels of these and other oxylipins in spr2 tissues may help ad-dress this issue. Of course, our results also leave open the pos-sibility that JA and its derivatives are not required strictly formale fertility in tomato. This notion is consistent with recentstudies showing that the sterility of a JA-insensitive mutant oftomato results mainly from a defect in female, not male, repro-ductive development (Li et al., 2001).

METHODS

Plant Material and Growth Conditions

Tomato (Lycopersicon esculentum) cv Castlemart was used as the wild-type parent for all experiments involving comparison of wild-type andmutant plants. Tomato seedlings were grown in Jiffy peat pots (HummertInternational, Earth City, MO) in a growth chamber maintained under 17 hof light (200 E·m�2·s�1) at 28C and 7 h of dark at 18C. Seeds of Lyco-persicon pennellii (LA716) and the introgression lines (Eshed and Zamir,1994) were obtained from the C.M. Rick Tomato Genetic Resource Cen-ter (University of California at Davis).

The original spr2 mutant (line 572A; Howe and Ryan, 1999), which wasidentified in the 35S:Prosys genetic background, was backcrossed to cvCastlemart to generate an F2 population segregating for both 35S:Prosysand spr2. To identify spr2 homozygotes that lack the transgene, F2plants that exhibit a wound-nonresponsive phenotype were identified.Genomic DNA from these plants was subjected to PCR screening usinga primer set (5�-GCGGATCCGTGGAGATGACAAAGAGACTCC-3� and5�-GCGGATCCGAAGTTACTTTTCTAACGGGAGAC-3�) that specificallyamplifies a 200-bp fragment corresponding to 35S:Prosys and a 1-kbfragment corresponding to the endogenous Prosys gene. DNA gel blot

analysis was used to confirm the absence of the transgene in selectedspr2 homozygotes. One such line was used as the female parent forthree successive backcrosses to the wild type.

Wound- and Elicitor-Response Assays

The wound response of tomato plants was determined using a radial im-munodiffusion assay for the detection of PI-II accumulation in leaf tissue(Ryan, 1967; Trautman et al., 1971). Briefly, 16- to 18-day-old seedlingscontaining two expanded leaves and a third emerging leaf werewounded with a hemostat across the midrib of all leaflets (typically three)on the lower leaf. Three hours later, the same leaflets were woundedagain, proximal to the petiole. Wounded plants were incubated for 24 hunder standard growth conditions, after which the wounded leaf (localresponse) and an upper, unwounded leaf (systemic response) were har-vested separately for determination of PI-II protein levels. Feeding ofelicitors was performed as described by Lee and Howe (2003). Systeminand oligogalacturonic acid were obtained from C.A. Ryan (WashingtonState University). 12-Oxo-phytodienoic acid and 13(S)-hydroperoxy lino-lenic acid were purchased from Cayman Chemical (Ann Arbor, MI). Jas-monic acid (JA) and bestatin were purchased from Sigma. All com-pounds were diluted from stock solutions into sodium phosphate buffer(15 mM sodium phosphate, pH 6.5) before use.

Map-Based Cloning of Spr2

The general strategy for mapping Spr2 was similar to that used to mapthe tomato Def1 gene (Li et al., 2002a). A BC1 mapping population wasconstructed from a cross between spr2/spr2 (L. esculentum) and L. pen-nellii (LA716), followed by backcrossing of a resulting F1 plant to spr2/spr2 (L. esculentum). Use of a BC1 population ensured that all segregat-ing progeny contained at least one copy of the L. esculentum PI-II gene,the product of which was used to generate the antibody used for the ra-dial immunodiffusion assay of PI-II. The wound-response phenotype ofindividual plants in the BC1 population was assessed quantitatively bycomparison with the wound response of wild-type and spr2 plantsgrown together with the BC1 plants. Plants showing an unambiguousphenotype were repotted and grown in the greenhouse for severalweeks, at which time leaf material was harvested for DNA extraction asdescribed by McCouch et al. (1988).

Using the BC1 population described above, bulked segregant analysiswas used in combination with amplified fragment length polymorphism(AFLP) analysis to identify molecular markers linked to Spr2. Equalamounts of DNA from 10 randomly selected wound-responsive (i.e.,wild-type) and 10 mutant BC1 plants were pooled to construct a wild-type bulk (B�) and a mutant bulk (B�), respectively. The procedures ofVos et al. (1995) and Thomas et al. (1995) were used to identify AFLPsthat distinguished the B� and wild-type parental (L. pennellii) DNA fromthe B� and mutant parental (spr2/spr2) DNA. Among 64 primer combina-tions used to screen the bulks for AFLPs, one combination (E-AAG/M-CAG) generated a product of �200 bp that was present in DNA fromboth the wild-type parent (Spr2pen/spr2esc) and the B� but absent in DNAfrom the mutant parent (spr2esc/spr2esc) and the B� (data not shown). TheDNA fragment corresponding to this AFLP marker was designated AF1.

The polymorphic band corresponding to AF1 was excised from thepolyacrylamide gel and cloned into the pGEM-T Easy vector (Promega)as described previously (Li et al., 2002a). Linkage of an AF1-derived re-striction fragment length polymorphism (RFLP) marker to Spr2 was con-firmed using the 20 BC1 individuals used to construct the bulks; allwound-responsive wild-type plants were heterozygous for the marker,whereas all mutant plants were homozygous for the L. esculentum RFLPpattern (data not shown). The absence of recombination events in thispopulation of 20 BC1 plants demonstrated tight linkage between AF1

1658 The Plant Cell

and Spr2. Tomato introgression lines (ILs) harboring defined chromo-some segments of L. pennellii in an L. esculentum background (Eshedand Zamir, 1994) were used to map AF1. Probing of survey blots con-taining L. esculentum and L. pennellii DNA showed that restriction en-zyme DraI generated a readily detectable RFLP using a 32P-labeled AF1probe. This RFLP allowed us to map AF1 to the IL6-1 region located be-tween RFLP markers TG612 and TG352 on chromosome 6, which is de-fined by IL LA3500.

Analysis of linkage between Spr2 and known RFLP markers on IL6-1(Tanksley et al., 1992) demonstrated that Spr2 is located between TG590and TG118. A high-resolution genetic map of the Spr2 region was con-structed by scoring 1436 BC1 plants for recombination events within theTG590-Spr2-TG118 interval. Results obtained from these experimentsplaced Spr2 �2.6 map units (38 recombination events) from TG118 and0.07 map units (one recombination event) from TG590. Three yeast artificialchromosome (YAC) end-derived markers, 258SL, 149AR, and 328N1,which were mapped previously in the TG590-TG118 interval (Ling et al.,2002), also were mapped relative to Spr2 (Figure 2). The mapping datashowed that 258SL cosegregated with Spr2 in all 1436 BC1 plants,whereas 149AR and 328N1 were located one and three recombinationevents, respectively, distant of Spr2.

Markers TG590, 258SL, and 149AR were used individually to screen atomato BAC library (Budiman et al., 2000; Ling et al., 2002). Restrictionenzyme digests were used to “fingerprint” the genomic inserts of 25BAC clones, prepared using the procedure of Budiman et al. (2000).Thermal asymmetric interlaced (TAIL) PCR (Liu and Whittier, 1995) wasused to obtain markers from the ends of eight selected BAC clones.These markers were mapped relative to Spr2 using recombination eventsin the TG590-TG118 interval. BAC end markers also were used to orderthe BAC clones relative to each other and to YAC149, resulting in con-struction of a contig spanning the Spr2 region (Figure 2). YAC149 andmarkers derived from it were described previously (Ling et al., 1996,2002). The left end of BAC101L7, named 101L7L, mapped one recombi-nation event away from Spr2. The right end of this BAC, 101L7R1, couldnot be mapped because of the presence of repetitive sequences. An ad-ditional round of TAIL-PCR using sequence information obtained from101L7R1, however, yielded a second marker, designated 101L7R2, onthe right end of BAC101L7. The sequences of the three 101L7R1-spe-cific primers used for this TAIL-PCR procedure were as follows: A,5�-GTGAGCGAACCAACCAATCTA-3�; B, 5�-GGAAGTCATCATCACAAG-AAC-3�; and C, 5�-AGCTAGTCCATGCCAGAACTT-3�. Mapping experi-ments indicated that 101L7R2 is located one recombination event awayfrom Spr2, on the opposite side of 101L7L. This information strongly in-dicated that Spr2 is located on BAC101L7.

DNA Sequence Analysis

DNA sequencing of tomato BAC101L7 was performed using a shotgunsequencing service provided by MWG-Biotech (High Point, NC). The as-sembled sequence consisted of seven contigs with a mean length of 16kb and totaling 114 kb. The largest contig (43,170 bp) contained theSpr2/LeFad7 gene as determined by Basic Local Alignment Search Tool(BLAST) searches against GenBank and the tomato EST database. Thesequence of this contig was deposited in GenBank. Oligonucleotideprimers GPB and GPb (see below) were designed to amplify LeFad7from wild-type and spr2 genomic DNA. The same primer set was used toobtain full-length LeFad7 cDNA clones using reverse transcription PCRof RNA isolated from wild-type and spr2 flowers. PCR-amplified frag-ments were cloned into the pGEM-T Easy plasmid vector. DNA se-quencing of genomic and cDNA clones was performed at the MichiganState University Genomics Technology Support Facility. The sequenceof the full-length LeFad7 cDNA obtained by reverse transcription PCRusing RNA from wild-type plants was deposited in GenBank.

Agrobacterium tumefaciens–Mediated Transformation

A 2.05-kb genomic fragment containing the Spr2/LeFad7 gene was ampli-fied from wild-type genomic DNA by PCR using the primer pair GPB(5�-ATCCCGGGTTCACCTAACCCAACATCTCATCT-3�) and GPb (5�-AAG-AGCTCTGCCTTCCCCCATCTTCTA-3�). This fragment was cloned intothe SmaI and SstI sites of the binary vector pBI121 (Clontech, Palo Alto,CA) under the control of the 35S promoter of Cauliflower mosaic virus. Theresulting construct was transformed into Agrobacterium tumefaciens strainAGLO (Lazo et al., 1991). Agrobacterium-mediated transformation of spr2cotyledon explants was performed as described previously (Li and Howe,2001). Regenerated kanamycin-resistant transformants in which expres-sion of the 35S:LeFad7 transgene was confirmed were potted into stan-dard soil mix and grown in a growth chamber under standard conditions.Approximately 3 weeks after transfer to soil, these plants were used for as-says of wound-induced PI-II accumulation and leaf fatty acid composition.

Quantification of JA

An approximately equal number of mature unopened flower buds (�6mm long) and newly opened flowers were harvested from wild-type andspr2 plants grown side by side in a greenhouse. A quantity of flowerscorresponding to 3 g fresh weight of tissue was used for each JA extrac-tion, performed as described by Li et al. (2002b). Levels of JA in tissueextracts were quantified by gas chromatography–mass spectrometryusing dihydro-JA as an internal standard (Li et al., 2002b). Extraction andquantification of JA was performed from three independent tissue sam-ples from each plant genotype.

Fatty Acid and Nucleic Acid Analysis

Tomato leaf and root tissue was harvested from 16- to 18-day-old plantsand frozen immediately in liquid nitrogen. Lipids were extracted as de-scribed by Dörmann et al. (1995). For quantitative analysis, individual lip-ids were separated by thin layer chromatography, scraped from theplates, and used to prepare fatty acid methyl esters (Xu et al., 2002). Therelative proportions of methyl ester fatty acids were quantified by gaschromatography as described by Xu et al. (2002).

Total RNA was isolated from tomato tissues and analyzed by gel blothybridization as described previously (Howe et al., 1996). Five microgramsof total RNA also was electrophoresed and stained with ethidium bromideto ensure equal loading of samples and intactness of the RNA. RNA gelswere blotted to Hybond-N� filters that were hybridized subsequently to a32P-labeled cDNA for PI-II (Graham et al., 1985) and LeFad7. As an addi-tional control for RNA loading, blots were hybridized to a cDNA probe (to-mato EST clone cLED1D24) for translation initiation factor eIF4A. DNA gelblot analysis was performed as described by Li et al. (2002a).

Insect Feeding Trials

Tobacco hornworm (Manduca sexta) eggs and artificial diet for horn-worm larvae were obtained from the Carolina Biological Supply Com-pany (Burlington, NC). Eggs were hatched at 26C as recommended bythe supplier. Hatched larvae were reared on the artificial diet for 3 daysbefore transfer to tomato plants. The average weight of larvae at the be-ginning of the feeding trial was �5 mg.

Upon request, all novel materials described in this article will be madeavailable in a timely manner for noncommercial research purposes.

Accession Numbers

The GenBank accession number for the contig containing the Spr2/LeFad7 gene is AY248742. The accession number for the full-length

Role of Spr2 in Systemin Signaling 1659

LeFad7 cDNA is AY248741. Accession numbers for the sequencesshown in Figure 4 are as follows: AtFAD3, AAL36322; AtFAD7,BAA03106; AtFAD8, AAA65621; StFAD7, T07685; OsFAD3, T03923;TaFAD3, T06238; TaFAD7, BAA07785; ZmFAD7, BAA22442; ZmFAD8,BAA22440; GmFAD3, BAB18135; GmFAD7, P48621; NtFAD3, P48623;NtFAD7, BAA11475; PfFAD7, AAB39387; SiFAD7, P48620; LeFAD7,AY248741; and putative FAD3, TC99736.

ACKNOWLEDGMENTS

We gratefully acknowledge Mike Pollard and Xiaoming Bao for theirhelpful assistance with fatty acid analysis, Lei Li for assistance with RNAgel blot experiments, Liyan Liu for assistance with RFLP mapping, andBonnie McCaig for help with phylogenetic analysis. We thank SteveTanksley for providing the tomato RFLP markers used in this study andClarence Ryan for providing systemin and OGA. The tomato BAC libraryand the tomato EST clone cLED1D24 were obtained from the ClemsonUniversity Genomics Institute. This research was supported by NationalInstitutes of Health Grant GM57795 and U.S. Department of EnergyGrant DE-FG02-91ER20021 to G.A.H.

Received March 22, 2003; accepted April 17, 2003.

REFERENCES

Allen, K.D. (2002). Assaying gene content in Arabidopsis. Proc. Natl.Acad. Sci. USA 99, 9568–9572.

Altschul, S.F., Gish, W., Miller, W., Myers, E.W., and Lipman, D.J.(1990). Basic local alignment search tool. J. Mol. Biol. 215, 403–410.

Bate, N.J., and Rothstein, S.J. (1998). C6-volatiles derived from thelipoxygenase pathway induce a subset of defense-related genes.Plant J. 16, 561–569.

Baydoun, E.A.H., and Fry, S.C. (1988). The immobility of pectic sub-stances in injured tomato leaves and its bearing on the identity of thewound hormone. Planta 165, 269–276.

Bishop, P.D., Makus, D.J., Pearce, G., and Ryan, C.A. (1981). Protein-ase inhibitor-inducing factor activity in tomato leaves resides in oli-gosaccharides enzymically released from cell walls. Proc. Natl. Acad.Sci. USA 78, 3536–3540.

Blechert, S., Bockelmann, C., Füsslein, M., Schrader, T.V., Stelmach,S.B., Niesel, U., and Weiler, E.W. (1999). Structure-activity analysesreveal the existence of two separate groups of active octadecanoidsin elicitation of the tendril-coiling response of Bryonia dioica Jacq.Planta 207, 470–479.

Blechert, S., Brodschelm, W., Holder, S., Kammerer, L., Kutchan,T.M., Mueller, M.J., Xia, Z.-Q., and Zenk, M.H. (1995). The octadeca-noic pathway: Signal molecules for the regulation of secondary path-ways. Proc. Natl. Acad. Sci. USA 92, 4099–4105.

Browse, J., McCourt, P., and Somerville, C.R. (1986). A mutant ofArabidopsis deficient in C18:3 and C16:3 leaf lipids. Plant Physiol. 81,859–864.

Browse, J., and Somerville, C. (1991). Glycerolipid synthesis: Bio-chemistry and regulation. Annu. Rev. Plant Physiol. Plant Mol. Biol.42, 467–506.

Budiman, M.A., Mao, L., Wood, T.C., and Wing, R.A. (2000). A deep-coverage tomato BAC library and prospects towards developmentof an STC framework for genome sequencing. Genome Res. 10,129–136.

Conconi, C., Miquel, M., Browse, J.A., and Ryan, C.A. (1996). Intra-cellular levels of free linolenic and linoleic acids increase in tomatoleaves in response to wounding. Plant Physiol. 111, 797–803.

Constabel, C.P., Yip, L., and Ryan, C.A. (1998). Prosystemin from

potato, black nightshade, and bell pepper: Primary structure and bio-logical activity of predicted systemin polypeptides. Plant Mol. Biol.36, 55–62.

Doares, S.H., Syrovets, T., Weiler, E.W., and Ryan, C.A. (1995). Oli-gogalacturonides and chitosan activate plant defense genes throughthe octadecanoid pathway. Proc. Natl. Acad. Sci. USA 92, 4095–4098.

Dörmann, P., Hoffmann-Benning, S., Balbo, I., and Benning, C.(1995). Isolation and characterization of an Arabidopsis mutant defi-cient in the thylakoid lipid digalactosyl diacylglycerol. Plant Cell 7,1801–1810.

Eshed, Y., and Zamir, D. (1994). A genomic library of Lycopersiconpennellii in L. esculentum: A tool for fine mapping of genes. Euphytica79, 175–179.

Farag, M.A., and Paré, P.W. (2002). C6-green leaf volatiles trigger localand systemic VOC emissions in tomato. Phytochemistry 61, 545–554.

Farmer, E.E., and Ryan, C.A. (1992). Octadecanoid precursors of jas-monic acid activate the synthesis of wound-inducible proteinaseinhibitors. Plant Cell 4, 129–134.

Felix, G., and Boller, T. (1995). Systemin induces rapid ion fluxes andethylene biosynthesis in Lycopersicon peruvianum cells. Plant J. 7,381–389.

Gatehouse, J.A. (2002). Plant resistance towards insect herbivores: Adynamic interaction. New Phytol. 156, 145–169.

Gibson, S., Arondel, V., Iba, K., and Somerville, C. (1994). Cloning of atemperature-regulated gene encoding a chloroplast �-3 desaturasefrom Arabidopsis thaliana. Plant Physiol. 106, 1615–1621.

Graham, J.S., Pearce, G., Merryweather, J., Titani, K., Ericsson,L.H., and Ryan, C.A. (1985). Wound-induced proteinase inhibitorsfrom tomato leaves. II. The cDNA-deduced primary structure of pre-inhibitor II. J. Biol. Chem. 260, 6561–6564.

Green, T.R., and Ryan, C.A. (1972). Wound inducible proteinase inhibi-tors in plant leaves: A possible defense mechanism against insects.Science 175, 776–777.

Hamada, T., Nishiuchi, T., Kodama, H., Nishimura, M., and Iba, K.(1996). cDNA cloning of a wounding-inducible gene encoding a plas-tid �3 fatty acid desaturase from tobacco. Plant Cell Physiol. 37,606–611.

Hause, B., Stenzel, I., Miersch, O., Maucher, H., Kramell, R., Ziegler,J., and Wasternack, C. (2000). Tissue-specific oxylipin signature oftomato flowers: Allene oxide cyclase is highly expressed in distinctflower organs and vascular bundles. Plant J. 24, 113–126.

Hilleren, P., and Parker, R. (1999). Mechanisms of mRNA surveillancein eukaryotes. Annu. Rev. Genet. 33, 229–260.

Howe, G.A., Lee, G.I., Itoh, A., Li, L., and DeRocher, A. (2000). Cyto-chrome P450-dependent metabolism of oxylipins in tomato: Cloningand expression of allene oxide synthase and fatty acid hydroperoxidelyase. Plant Physiol. 123, 711–724.

Howe, G.A., Lightner, J., Browse, J., and Ryan, C.A. (1996). An octa-decanoid pathway mutant (JL5) of tomato is compromised in signal-ing for defense against insect attack. Plant Cell 8, 2067–2077.

Howe, G.A., and Ryan, C.A. (1999). Suppressors of systemin signalingidentify genes in the tomato wound response pathway. Genetics 153,1411–1421.

Hugly, S., and Somerville, C.R. (1992). A role for membrane lipid poly-unsaturation in chloroplast biogenesis at low temperature. PlantPhysiol. 99, 197–202.

Iba, K., Gibson, S., Nishiuchi, T., Fuse, T., Nishimura, M., Arondel, V.,Hugly, S., and Somerville, C. (1993). A gene encoding a chloroplast�-3 fatty acid desaturase complements alterations in fatty acid desat-uration and chloroplast copy number of the fad7 mutant of Arabidop-sis thaliana. J. Biol. Chem. 268, 24099–24105.

Ishiguro, S., Kawai-Oda, A., Ueda, K., Nishida, I., and Okada, K.

1660 The Plant Cell

(2001). The DEFECTIVE IN ANTHER DEHISCENCE1 gene encodes anovel phospholipase A1 catalyzing the initial step of jasmonic acidbiosynthesis, which synchronizes pollen maturation, anther dehis-cence, and flower opening in Arabidopsis. Plant Cell 13, 2191–2209.

Jaglo, K.R., Kleff, S., Amundsen, K.L., Zhang, X., Haake, V., Zhang,J.Z., Deits, T., and Thomashow, M.F. (2001). Components of theArabidopsis C-repeat/dehydration-responsive element binding factorcold-response pathway are conserved in Brassica napus and otherplant species. Plant Physiol. 127, 910–917.

Jamieson, G.R., and Reid, E.H. (1971). The occurrence of hexadeca-7,10,13-trienoic acid in the leaf lipids of angiosperms. Phytochemistry10, 1837–1843.

Karban, R., and Baldwin, I.T. (1997). Induced Response to Herbivory.(Chicago, IL: University of Chicago Press).

Keegstra, K., Olesen, L.J., and Theg, S.M. (1989). Chloroplastic pre-cursors and their transport across the envelope membranes. Annu.Rev. Plant Physiol. Plant Mol. Biol. 40, 471–501.

Kessler, A., and Baldwin, I.T. (2002). Plant responses to insect her-bivory: The emerging molecular analysis. Annu. Rev. Plant Biol. 53,299–328.

Kubigsteltig, I., Laudert, D., and Weiler, E.W. (1999). Structure andregulation of the Arabidopsis thaliana allene oxide synthase gene.Planta 208, 463–471.

Kwon, J.H., Lee, Y.M., and An, C.S. (2000). cDNA cloning of chloro-plast omega-3 fatty acid desaturase from Capsicum annuum and itsexpression upon wounding. Mol. Cells 10, 493–497.

Lazo, G.R., Stein, P.A., and Ludwig, R.A. (1991). A DNA transforma-tion-competent Arabidopsis genomic library in Agrobacterium. Bio-technology 9, 963–967.

LeBrasseur, N.D., MacIntosh, G.C., Perez-Amador, M.A., Saitoh, M.,and Green, P.J. (2002). Local and systemic wound-induction ofRNase and nuclease activities in Arabidopsis: RNS1 as a marker for aJA-independent systemic signaling pathway. Plant J. 29, 393–403.

Lee, G.I., and Howe, G.A. (2003). The tomato mutant spr1 is defectivein systemin perception and the production of a systemic wound sig-nal for defense gene expression. Plant J. 33, 567–576.

León, J., Rojo, E., and Sánchez-Serrano, J.J. (2001). Wound signalingin plants. J. Exp. Bot. 52, 1–9.

Li, C., Williams, M.M., Loh, Y.-T., Lee, I.G., and Howe, G.A. (2002a).Resistance of cultivated tomato to cell content-feeding herbivores isregulated by the octadecanoid-signaling pathway. Plant Physiol. 130,494–503.

Li, L., and Howe, G.A. (2001). Alternative splicing of prosystemin pre-mRNA produces two isoforms that are active as signals in the woundresponse pathway. Plant Mol. Biol. 46, 409–419.

Li, L., Li, C., and Howe, G.A. (2001). Genetic analysis of wound signal-ing in tomato: Evidence for a dual role of jasmonic acid in defenseand female fertility. Plant Physiol. 127, 1414–1417.

Li, L., Li, C., Lee, G.I., and Howe, G.A. (2002b). Distinct roles for jas-monic acid synthesis and action in the systemic wound response oftomato. Proc. Natl. Acad. Sci. USA 99, 6416–6421.

Liechti, R., and Farmer, E.E. (2002). The jasmonate pathway. Science296, 1649–1650.

Ling, H.-Q., Bauer, P., Bereczky, Z., Keller, B., and Ganal, M. (2002).The tomato fer gene encoding a bHLH protein controls iron-uptakeresponses in roots. Proc. Natl. Acad. Sci. USA 99, 13938–13943.

Ling, H.Q., Pich, A., Scholz, G., and Ganal, M.W. (1996). Genetic anal-ysis of two tomato mutants affected in the regulation of iron metabo-lism. Mol. Gen. Genet. 252, 87–92.

Liu, Y.G., and Whittier, R.F. (1995). Thermal asymmetric interlacedPCR-automatable amplification and sequencing of insert end frag-ments from P1 and YAC clones for chromosome walking. Genomics25, 674–681.

Malone, M. (1996). Rapid, long-distance signal transmission in higherplants. Adv. Bot. Res. 22, 163–228.

Martín, M., León, J., Dammann, C., Albar, J.-P., Griffiths, G., andSerrano, J.J.S. (1999). Antisense-mediated depletion of potato leaf�3 fatty acid desaturase lowers linolenic acid content and reducesgene activation in response to wounding. Eur. J. Biochem. 262,283–290.

McConn, M., and Browse, J. (1996). The critical requirement for lino-lenic acid is pollen development, not photosynthesis, in an Arabidop-sis mutant. Plant Cell 8, 403–416.

McConn, M., Hugly, S., Browse, J., and Somerville, C. (1994). Amutation at the fad8 locus of Arabidopsis identifies a second chloro-plast �3 desaturase. Plant Physiol. 106, 1609–1614.

McCouch, S.R., Kochert, G., Yu, Z.H., Wang, Z.Y., Khush, G.S.,Coffman, W.R., and Tanksley, S.D. (1988). Molecular mapping ofrice chromosomes. Theor. Appl. Genet. 76, 815–829.

McGurl, B., Orozco-Cardenas, M., Pearce, G., and Ryan, C.A. (1994).Overexpression of the prosystemin gene in transgenic tomato plantsgenerates a systemic signal that constitutively induces proteinaseinhibitor synthesis. Proc. Natl. Acad. Sci. USA 91, 9799–9802.

McGurl, B., Pearce, G., Orozco-Cardenas, M., and Ryan, C.A. (1992).Structure, expression, and antisense inhibition of the systemin pre-cursor gene. Science 255, 1570–1573.

Meindl, T., Boller, T., and Felix, G. (1998). The plant wound hormonesystemin binds with the N-terminal part to its receptor but needs theC-terminal part to activate it. Plant Cell 10, 1561–1570.

Miquel, M., James, D.J., Dooner, H., and Browse, J. (1993). Arabi-dopsis requires polyunsaturated lipids for low-temperature survival.Proc. Natl. Acad. Sci. USA 90, 6208–6212.

Moyen, C., Hammond-Kosack, K.E., Jones, J., Knight, M.R., andJohannes, E. (1998). Systemin triggers an increase of cytoplasmiccalcium in tomato mesophyll cells: Ca2� mobilization from intra- andextracellular compartments. Plant Cell Environ. 21, 1101–1111.

Moyen, C., and Johannes, E. (1996). Systemin transiently depolarizesthe tomato mesophyll cell membrane and antagonizes fusicoccin-induced extracellular acidification of mesophyll tissue. Plant CellEnviron. 19, 464–470.

Narváez-Vásquez, J., Florin-Christensen, J., and Ryan, C.A. (1999).Positional specificity of a phospholipase A activity induced bywounding, systemin, and oligosaccharide elicitors in tomato leaves.Plant Cell 11, 2249–2260.

Nishiuchi, T., Hamada, T., Kodoma, H., and Iba, K. (1997). Woundingchanges the spatial expression pattern of the Arabidopsis plastidomega-3 fatty acid desaturase gene (FAD7) through different signaltransduction pathways. Plant Cell 9, 1701–1712.

Novitskaya, G.V., Suvorova, T.A., and Trunova, T.I. (2000). Lipid com-position of tomato leaves as related to plant cold tolerance. RussianJ. Plant Physiol. 47, 728–733.

O’Donnell, P.J., Calvert, C., Atzorn, R., Wasternack, C., Leyser,H.M.O., and Bowles, D.J. (1996). Ethylene as a signal mediating thewound response of tomato plants. Science 274, 1914–1917.

O’Donnell, P.J., Truesdale, M.R., Calvert, C.M., Dorans, A.,Roberts, M.R., and Bowles, D.J. (1998). A novel tomato gene thatrapidly responds to wound- and pathogen-related signals. Plant J.14, 137–142.

Orozco-Cárdenas, M., McGurl, B., and Ryan, C.A. (1993). Expressionof an antisense prosystemin gene in tomato plants reduces resis-tance toward Manduca sexta larva. Proc. Natl. Acad. Sci. USA 90,8273–8276.

Orozco-Cárdenas, M.L., Narváez-Vásquez, J., and Ryan, C.A.(2001). Hydrogen peroxide acts as a second messenger for theinduction of defense genes in tomato plants in response to wounding,systemin, and methyl jasmonate. Plant Cell 13, 179–191.

Role of Spr2 in Systemin Signaling 1661

Ouariti, O., Boussama, N., Zarrouk, M., Cherif, A., and Ghorbal, M.H.(1997). Cadmium- and copper-induced changes in tomato membranelipids. Phytochemistry 45, 1343–1350.

Pearce, G., Moura, D.S., Stratmann, J., and Ryan, C.A. (2001). Pro-duction of multiple plant hormones from a single polyprotein precur-sor. Nature 411, 817–820.

Pearce, G., Strydom, D., Johnson, S., and Ryan, C.A. (1991). Apolypeptide from tomato leaves induces wound-inducible proteinaseinhibitor proteins. Science 253, 895–898.

Rojo, E., León, J., and Sánchez-Serrano, J.J. (1999). Cross-talkbetween wound signalling pathways determines local versus sys-temic gene expression in Arabidopsis thaliana. Plant J. 20, 135–142.

Roughan, P.G., Holland, R., and Slack, C.R. (1980). The role of chlo-roplasts and microsomal fractions in polar-lipid synthesis from[1-14C]acetate by cell-free preparations from spinach (Spinacia olera-cea) chloroplasts. Biochem. J. 188, 17–24.

Routaboul, J.M., Fischer, S.F., and Browse, J. (2000). Trienoic fattyacids are required to maintain chloroplast function at low tempera-tures. Plant Physiol. 124, 1697–1705.

Ryan, C.A. (1967). Quantitative determination of soluble cellular pro-teins by radial diffusion in agar gels containing antibodies. Anal. Bio-chem. 19, 434–440.

Ryan, C.A. (2000). The systemin signaling pathway: Differential activa-tion of plant defensive genes. Biochim. Biophys. Acta 1477, 112–121.

Ryan, C.A., and Moura, D.S. (2002). Systemic wound signaling inplants: A new perception. Proc. Natl. Acad. Sci. USA 99, 6519–6520.

Ryan, C.A., Pearce, G., Scheer, J., and Moura, D.S. (2002). Polypep-tide hormones. Plant Cell 14 (suppl.), S251–S264.

Schaller, A., Bergey, D.R., and Ryan, C.A. (1995). Induction of woundresponse genes in tomato leaves by bestatin, an inhibitor of ami-nopeptidases. Plant Cell 7, 1893–1898.

Schaller, A., and Oecking, C. (1999). Modulation of plasma membraneH�-ATPase activity differentially activates wound and pathogendefense responses in tomato plants. Plant Cell 11, 263–272.

Scheer, J.M., and Ryan, C.A. (1999). A 160-kD systemin receptor onthe surface of Lycopersicon peruvianum suspension-cultured cells.Plant Cell 11, 1525–1536.

Scheer, J.M., and Ryan, C.A. (2002). The systemin receptor SR160from Lycopersicon peruvianum is a member of the LRR receptorkinase family. Proc. Natl. Acad. Sci. USA 99, 9585–9590.

Somerville, C.R., and Browse, J. (1991). Plant lipids: Metabolism,mutants and membranes. Science 252, 82–87.

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.

Strassner, J., Schaller, F., Frick, U.B., Howe, G.A., Weiler, E.W.,Amrhein, N., Macheroux, P., and Schaller, A. (2002). Characteriza-tion and cDNA-microarray expression analysis of 12-oxophytodi-enoate reductases reveals differential roles for octadecanoid biosyn-thesis in the local versus the systemic wound response. Plant J. 32,585–601.

Stratmann, J.W., and Ryan, C.A. (1997). Myelin basic protein kinaseactivity in tomato leaves is induced systemically by wounding andincreases in response to systemin and oligosaccharide elicitors. Proc.Natl. Acad. Sci. USA 94, 11085–11089.

Tanksley, S.D., et al. (1992). High density molecular linkage maps ofthe tomato and potato genomes. Genetics 132, 1141–1160.

Thomas, C.M., Vos, P., Zabeau, M., Jones, D.A., Norcott, K.A.,Chadwick, B.P., and Jones, J.D.G. (1995). Identification of amplifiedrestriction fragment polymorphism (AFLP) markers tightly linked tothe tomato Cf-9 gene for resistance to Cladosporium fulvum. Plant J.8, 785–794.

Titarenko, E., Rojo, E., León, J., and Sánchez-Serrano, J.J. (1997).Jasmonic acid-dependent and -independent signaling pathways con-trol wound-induced gene activation in Arabidopsis thaliana. PlantPhysiol. 11, 817–826.

Trautman, R., Cowan, K.M., and Wagner, C.G. (1971). Data process-ing for radial immunodiffusion. Immunochemistry 8, 901–916.

Turner, J.G., Ellis, C., and Devoto, A. (2002). The jasmonate signalpathway. Plant Cell 14 (suppl.), S153–S164.

Vancanneyt, G., Sanz, C., Farmaki, T., Paneque, M., and Ortego, F.(2001). Hydroperoxide lyase depletion in transgenic potato plantsleads to an increase in aphid performance. Proc. Natl. Acad. Sci. USA98, 8139–8144.

Van der Hoeven, R., Ronning, C., Giovannoni, J., Martin, G., andTanksley, S. (2002). Deductions about the number, organization, andevolution of genes in the tomato genome based on analysis of a largeexpressed sequence tag collection and selective genomic sequenc-ing. Plant Cell 14, 1441–1456.

Vos, P., Hogers, R., Bleeker, M., Reijans, M., van de Lee, T., Hornes,M., Frijters, A., Pot, J., Peleman, J., Kuiper, M., and Zabeau, M.(1995). AFLP: A new technique for DNA finger printing. Nucleic AcidsRes. 23, 4407–4414.

Walling, L.L. (2000). The myriad plant responses to herbivores. J. PlantGrowth Regul. 19, 195–216.

Wallis, J.G., and Browse, J. (2002). Mutants of Arabidopsis revealmany roles for membrane lipids. Prog. Lipid Res. 41, 254–278.

Wasternack, C., and Hause, B. (2002). Jasmonates and octa-decanoids: Signals in plant stress responses and development. Prog.Nucleic Acid Res. Mol. Biol. 72, 165–221.

Wasternack, C., Ortel, B., Miersch, O., Kramell, R., Beale, M.,Greulich, F., Feussner, I., Hause, B., Krumm, T., Boland, W., andParthier, B. (1998). Diversity in octadecanoid-induced gene expres-sion in tomato. J. Plant Physiol. 152, 345–352.

Weber, H. (2002). Fatty acid-derived signals in plants. Trends Plant Sci.7, 217–224.

Weber, H., Vick, B.A., and Farmer, E.E. (1997). Dinor-oxo-phytodi-enoic acid: A new hexadecanoid signal in the jasmonate family. Proc.Natl. Acad. Sci. USA 94, 10473–10478.

Whitaker, B.D. (1986). Fatty-acid composition of polar lipids in fruitand leaf chloroplasts of “16:3” and “18:3” plant species. Planta 169,313–319.

Xu, C., Härtel, H., Wada, H., Hagio, M., Yu, B., Eakin, C., andBenning, C. (2002). The pgp1 mutant locus of Arabidopsis encodes aphosphatidylglycerolphosphate synthase with impaired activity. PlantPhysiol. 129, 594–604.

Yang, Y.W., Lai, K.N., Tai, P.Y., and Li, W.H. (1999). Rates of nucle-otide substitution in angiosperm mitochondrial DNA sequences anddates of divergence between Brassica and other angiosperm lin-eages. J. Mol. Evol. 48, 597–604.

DOI 10.1105/tpc.012237; originally published online May 29, 2003; 2003;15;1646-1661Plant Cell

Ganal and Gregg A. HoweChuanyou Li, Guanghui Liu, Changcheng Xu, Gyu In Lee, Petra Bauer, Hong-Qing Ling, Martin W.

for Defense Gene ExpressionRequired for the Biosynthesis of Jasmonic Acid and the Production of a Systemic Wound Signal

Gene Encodes a Fatty Acid DesaturaseSuppressor of prosystemin-mediated responses2The Tomato

 This information is current as of June 24, 2020

 

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