hormonal and stress induction of the gene encoding · et al., 1998), but, after wounding, an...

Hormonal and Stress Induction of the Gene EncodingCommon Bean Acetyl-Coenzyme A Carboxylase1[W]

Rosa Elia Figueroa-Balderas, Berenice Garcıa-Ponce2, and Mario Rocha-Sosa*

Plant Molecular Biology, Instituto de Biotecnologia, Universidad Nacional Autonoma de Mexico,Cuernavaca, Morelos 62250, Mexico

Regulation of the cytosolic acetyl-coenzyme A carboxylase (ACCase) gene promoter from common bean (Phaseolus vulgaris)was studied in transgenic Arabidopsis (Arabidopsis thaliana) plants using a b-glucuronidase (GUS) reporter gene fusion(PvACCaseTGUS). Under normal growth conditions, GUS was expressed in hydathodes, stipules, trichome bases, flowers,pollen, and embryos. In roots, expression was observed in the tip, elongation zone, hypocotyl-root transition zone, and lateralroot primordia. The PvACCase promoter was induced by wounding, Pseudomonas syringae infection, hydrogen peroxide,jasmonic acid (JA), ethylene, or auxin treatment. Analysis of PvACCaseTGUS expression in JA and ethylene mutants(coronatine insensitive1-1 [coi1-1], ethylene resistant1-1 [etr1-1], coi1-1/etr1-1) suggests that neither JA nor ethylene perceptionparticipates in the activation of this gene in response to wounding, although each of these independent signaling pathways issufficient for pathogen or hydrogen peroxide-induced PvACCase gene expression. We propose a model involving differentpathways of PvACCase gene activation in response to stress.

Acetyl-CoA carboxylase (ACCase; EC 6.4.1.2) cata-lyzes the ATP-dependent formation of malonyl-CoAfrom acetyl-CoA and bicarbonate. ACCase and itsproduct, malonyl-CoA, play a key role in both primaryand secondary metabolism. In plastids, malonyl-CoAis essential for fatty acid synthesis (Harwood, 1988);cytosolic malonyl-CoA is required for flavonoid, stil-bene, and very long-chain fatty acid (VLCFA) synthe-sis (Ebel and Hahlbrock, 1977; Schroder et al., 1988;Roesler et al., 1994), as well as for the malonylation of1-aminocyclopropane-1-carboxylic acid (ACC) andD-amino acids (Liu et al., 1983). Two physically distinctheteromeric and homodimeric forms are found inflowering plants. Most species have both forms withheteromeric enzymes in plastids and the homodimericenzyme in the cytosol. In grasses, the homodimericform is found in both the plastid and cytosol (Konishi

and Sasaki, 1994; Konishi et al., 1996) and no hetero-meric enzyme exists. Plastids of Brassica napus (Schulteet al., 1997) contain both a homodimeric and the ex-pected heteromeric isoform. This could also be truefor Arabidopsis (Arabidopsis thaliana) because oneof the two predicted homodimeric ACCase genes(ACC2) encodes a putative chloroplastic transitpeptide.

A recent study has shown that the cytosolic form ofACCase (ACC1) is essential for the survival of Arabi-dopsis. In this context, Baud et al. (2003) showed thatacc1 mutants displayed defects in embryo develop-ment and in VLCFA synthesis. Mutants in the GURKEgene, which encodes ACC1, exhibit embryo defectsand its cotyledons are absent or highly reduced(Kajiwara et al., 2004). Nonetheless, these phenotypeswere reduced by an exogenous supply of malonate,suggesting that cytosolic ACCase is essential for nor-mal embryo development (Baud et al., 2004).

A second key role for cytosolic ACCase is thesynthesis of flavonoids. Flavonoids can act as sun-screens against harmful UV-B irradiation, therebypreventing damage to photosynthetic organs (Loisand Buchanan, 1994; Landry et al., 1995). Indeed,only the cytosolic form of ACCase, but not the chlo-roplastic isoform, is induced by UV-B irradiation. Thisresponse results in higher malonyl-CoA concentra-tions for flavonoid synthesis in the cytosol (Konishiet al., 1996). Flavonoids have also been implicated asendogenous negative regulators of polar auxin trans-port (Brown et al., 2001) and are important phyto-alexins in legumes (Dixon and Pavia, 1995). Inaddition, it has been shown that flavonoids can pro-vide protection by acting as scavengers of reactiveoxygen species (ROS; Yamasaki et al., 1997).

1 This work was supported by Consejo Nacional de Ciencia yTecnologıa (grant no. 28052N) and Direccion General de Asuntos delPersonal Academico, Universidad Nacional Autonoma de Mexico(grants nos. IN212103 and IX215304). R.E.F.-B. was a recipient ofConsejo Nacional de Ciencia y Tecnologia and Direccion General deEstudios de Posgrado-Universidad Nacional Autonoma de Mexicofellowships.

2 Present address: Functional Ecology, Instituto de Ecologıa,Universidad Nacional Autonoma de Mexico, Ciudad Universitariac.p. 04510, Mexico City, D.F., Mexico.

* Corresponding author; e-mail [email protected]; fax 52–777–3172388.

The author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Mario Rocha-Sosa ([email protected]).

[W] The online version of this article contains Web-only data.www.plantphysiol.org/cgi/doi/10.1104/pp.106.085597

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A common bean (Phaseolus vulgaris) cytosolic ACC-ase (PvACCase) partial cDNA clone was previouslyreported (Garcıa-Ponce and Rocha-Sosa, 2000). mRNAaccumulation of the first enzymes of flavonoid bio-synthesis PvACCase and chalcone synthase (ChS) wasinduced by methyl jasmonate (MeJA), yeast (Saccharo-myces cerevisiae) elicitor, or Pseudomonas syringae pvtabaci treatment (Garcıa-Ponce and Rocha-Sosa, 2000).Collectively, these data suggest coordinate regulationof these two early steps in the flavonoid pathway.Selective inhibitor treatments led to the conclusionthat ethylene and oxylipins were necessary for theinduction of the PvACCase gene in response toP. syringae pv tabaci (Garcıa-Ponce, 2000; Garcıa-Ponceand Rocha-Sosa, 2000).

Oxylipins are oxidation products derived from fattyacids and they have both signaling and antimicrobialactivity. The best-studied oxylipin is jasmonic acid(JA). JA synthesis is induced in plants by wounding orpathogen attack, leading to the induction of a batteryof defense genes (Devoto et al., 2005). The precursor ofJA, 12-oxophytodienoic acid (OPDA), is induced afterwounding or elicitor treatment (Parchmann et al.,1997; Stintzi et al., 2001). In Arabidopsis, OPDA canactivate wound-induced gene expression in the ab-sence of JA (Taki et al., 2005). In parsley (Petroselinumcrispum), soybean (Glycine max), and bean, eitherOPDA or JA promotes the synthesis of flavonoids andthe expression of their biosynthetic genes (Franceschiand Grimes, 1991; Dittrich et al., 1992; Garcıa-Ponceand Rocha-Sosa, 2000). In addition to oxylipins, ethyl-ene has also been implicated in the activation ofsystemic plant defenses in response to pathogens(Penninckx et al., 1998) and wounding (O’Donnellet al., 1996). In Arabidopsis, ethylene also provokes anincrease in flavonoid accumulation (Buer et al., 2006)and in carrot (Daucus carota) induces genes for flavo-noid synthesis (Ecker and Davis, 1987). The JA andethylene signaling pathways are induced concomitantlyin Arabidopsis to activate defense responses afterinfection with a necrotrophic pathogen (Penninckxet al., 1998), but, after wounding, an antagonisticinteraction between JA and ethylene was proposedfor the local responses in Arabidopsis leaves (Rojoet al., 1999). In contrast, in tomato (Lycopersicon escu-lentum), ethylene potentiates JA action in response towounding (O’Donnell et al., 1996). Thus, plant re-sponses to pathogens and wounding are complex andparticipation of JA and ethylene varies.

Despite the importance of the cytosolic ACCaseenzyme in plants and its documented increase afterpathogen attack, to date, to our knowledge, there isno analysis of the tissue-specific expression of theACCase genes and its regulation by developmentaland environmental cues. To explore these facets ofACCase regulation, we analyzed expression in com-mon bean and we generated transgenic Arabidopsisplants expressing the b-glucuronidase (GUS) reportergene under the control of the common bean ACCasegene promoter. The patterns of PvACCase gene ex-

pression under normal growth or stress conditionswere analyzed. To determine the hierarchy between JAand ethylene signals, an analysis of PvACCase genepromoter activity in JA (coronatine insensitive1-1 [coi1-1])and ethylene (ethylene resistant1-1 [etr1-1]) perceptionmutants was also performed.

RESULTS

PvACCase mRNA Accumulation Is Induced

by Wounding and Ethylene in Common Bean

Previously, we analyzed the PvACCase mRNA ac-cumulation pattern in response to pathogen infectionand MeJA or elicitor treatment in cell cultures andleaves from common bean. As mentioned above, inaddition to being induced by MeJA in cell cultures andleaves, ACCase mRNA accumulated after yeast elici-tor or P. syringae pv tabaci treatment. Inhibitors of theoctadecanoid pathway severely reduced ACCase mRNAand protein accumulation induced by the yeast elicitoror P. syringae pv tabaci, indicating that jasmonates or aprecursor mediate ACCase induction after pathogeninfection (Garcıa-Ponce and Rocha-Sosa, 2000). Be-cause JA induces genes that respond to wounding, wehave now tested whether wounding affects PvACCasemRNA levels in bean leaves. A gene-specific probefor the 3#-untranslated region of the PvACCase genewas used in this expression analysis. PvACCasemRNA is detected 3 h after wounding and its levelremained almost constant 12 h after wounding (Fig.1A). Because ethylene has also been implicated in re-sponse to wounding or pathogen infection (O’Donnellet al., 1996; Penninckx et al., 1998), we treated commonbean plants with ethephon (an ethylene-releasingagent) to determinate whether PvACCase expressionis induced by ethylene. PvACCase mRNA was detec-ted 1 h after ethephon addition, reached a peak at 6 h,and decreased afterward (Fig. 1B). Considered to-gether with our previous data, these results suggestthat a JA- and ethylene-responsive signaling pathwayis necessary for the stress response of the PvACCasegene. In Arabidopsis, a JA/ethylene-mediated signal-ing pathway in response to wounding or pathogenattack has been described (Penninckx et al., 1998; Rojoet al., 1999). To analyze the role of JA and ethylene inthe stress response of the PvACCase gene moredeeply, we constructed Arabidopsis plants expressingthe GUS reporter gene under the control of thePvACCase gene promoter.

Cloning and Analysis of the Common Bean PvACCaseGene Promoter Region

Genomic libraries were prepared from commonbean DNA using the Universal Genome Walker kit(see ‘‘Materials and Methods’’). Four PvACCase genepromoter fragments (406, 486, 786, and 2,716 bp up-stream from the putative ATG start codon; data not

Figueroa-Balderas et al.

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shown) were sequenced and analyzed for regulatorymotifs and promoter elements. At high stringency,matches to cis-elements previously identified asmediators of pathogen responses were found inPvACCase gene promoters (Fig. 2). For example, theW1-box, a cis-element that binds WRKY transcriptionfactors (Eulgem et al., 1999), was found 288 bp up-stream of the ATG start codon. The G-box (21,092) andH-box (21,048) involved in tissue-specific expression

activation of the bean chs15 gene promoter (Faktoret al., 1997) were also found (Fig. 2). Other elementsidentified by promoter scanning using the PlantCAREdatabase (Lescot et al., 2002; http://bioinformatics.psb.ugent.be/webtools/plantcare/html) include theethylene response elements (22,609 and 22,426), theTGA box (21,292), and the CGTCA motifs (21,289,2968, and 2834), which are involved in auxin andMeJA responsiveness, respectively. Most of these ele-ments are conserved in sequence, but not in position inthe Arabidopsis and the soybean cytosolic ACCasegene promoters (Fig. 2).

Because the four DNA fragments from the putativecontrol region of the PvACCase gene represent adeletion series from the 5# end of the presumptivepromoter, they were each fused transcriptionally to theGUS reporter gene and used to transform Arabidopsisecotype Columbia-0 (Col-0). Homozygous T3 plantswere analyzed from three independent lines per con-struct. Only the construct containing 2.7 kb upstreamof the ATG start codon (PvACCase::GUS) was able tosupport detectable GUS activity (data not shown).Therefore, the minimal promoter is .786 bp long,surprisingly large for a plant gene; the motifs con-served in soybean and Arabidopsis extend to 2900and 22,500 bp, respectively.

Organ-Specific Expression of PvACCase::GUSGene Fusion

Tissue-specific expression of PvACCaseTGUSwas monitored by histochemical staining. GUS activitywas observed in hydathodes of young and adult leaves,

Figure 1. PvACCase mRNA expression is induced by wounding andethylene in common bean plants. A, PvACCase mRNA accumulation inresponse to wounding. Leaves of 13-d-old plants were wounded withdialysis closure clips for the indicated times. B, Kinetics of PvACCasetranscript accumulation following ethephon treatment. Plants weregrown for 23 d in a hydroponic system and treated with 100 mM

ethephon. Total RNA (30 mg/lane) was analyzed by hybridizing witha gene-specific probe for the 3#-untranslated region of PvACCase gene(AF007803). rRNA stained with ethidium bromide is shown as aloading control (bottom). A representative experiment of at least threeindependent experiments is shown.

Figure 2. Schematic comparison of the cytosolic ACCase promoters from common bean, soybean, and Arabidopsis. Thelocation of various putative cis-elements of interest was identified using the PLANTCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html). The numbers indicate the PvACCase gene promoter fragments used in detection analysis. Pv,Phaseolus vulgaris; Gm, Glycine max; and At, Arabidopsis.

PvACCase Wound-Induced Expression Is JA/Ethylene Independent

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stipules, stamens, stigma, pollen, siliques, embryos,and the base of some trichomes near the hydathodes(Fig. 3). In Figure 4, the expression pattern in roots of 3-,5-, and 7-d-old seedlings is shown. At 3 d, GUS activitywas observed in the whole root (Fig. 4A). At 5 and 7 d,GUS activity was detected only from the hypocotyl-root transition zone until the elongation zone. At theroot tip, staining was noticed in 5-d-old seedlings, butwas absent in 7-d-old seedlings (Fig. 4, B and C).Nonetheless, GUS activity was also detected at the sitesof lateral root formation in 7-d-old seedlings (Fig. 4, Eand F). By 14 d, secondary roots had developed andtheir GUS expression pattern was the same as that of theprimary root, with staining in the elongation zone androot tip (Fig. 4). Overall, this pattern of expression (highin roots and flowers, low in leaves) was in agreementwith the organ-specific accumulation of PvACCasemRNA in common bean plants (Fig. 5).

To confirm the results of the GUS analysis, whole-mount in situ hybridization was performed to localizeACCase mRNA. Gene-specific probes for ArabidopsisACC1 and PvACCase genes were expressed in roottissue of 3-d-old seedlings in the elongation zoneand root tip, as well as in the lateral root primordia(Fig. 6). No signal was observed when the AtACC1 andPvACCase sense probes were used. In addition, wealso observed accumulation of AtACC1 mRNA instipules and hydathodes (data not shown). Theseobservations confirm that GUS accumulation fromthe PvACCaseTGUS transgene reflects the in vivodistribution of transcripts from both the PvACCasegene and the endogenous ACC1 gene.

PvACCase Response to Exogenous Auxin Application

Auxins are inducers of lateral root formation(Boerjan et al., 1995; Casimiro et al., 2001) because ex-

ogenous application of indole acetic acid (IAA) stimu-lates lateral root production (Evans et al., 1994). It isnoteworthy that PvACCase::GUS transgene expres-sion during development has a strong correlation to thetissues where auxins are accumulated in Arabidopsis.For example, several studies have shown high auxinconcentrations in Arabidopsis hydathodes, stipules,and root tips; furthermore, flavonoids colocalize withzones of auxin accumulation (Murphy et al., 2000; Peeret al., 2001; Aloni et al., 2003). To determine auxinresponsiveness of the PvACCase gene promoter, 8-d-old roots were treated with IAA. At 6 h, the expressionpattern was unaffected; however, at 24 and 48 h, GUSexpression was more strongly activated in the initiatinglateral roots following treatment with IAA (Supple-mental Fig. S1). Based on these data, we suggest that thePvACCase gene promoter responds to a developmen-tal signal (lateral root formation) induced by auxinsand not directly to treatment with this phytohormone.Simultaneously, we analyzed the auxin effect on pro-moter induction in aerial tissues on PvACCaseTGUS8-d-old seedlings. As illustrated in Figure 8, auxintreatment resulted in a significant induction of thePvACCaseTGUS gene promoter in leaves.

Stress Activation

Wounding induces expression of Arabidopsis genesrequired in flavonoid metabolism (i.e. ChS and Pheammonia lyase; Reymond et al., 2000). We also dem-onstrated that wounding induced PvACCase mRNAaccumulation in common bean leaves (Fig. 1). To assaywounding responsiveness of the PvACCase gene pro-moter, 5- to 6-week-old leaves from PvACCase::GUSplants were mechanically wounded with dissectionforceps and analyzed for GUS activity. Within 6 h,strong local staining was evident surrounding the

Figure 3. Expression pattern of PvACCaseTGUS intransgenic Arabidopsis. A, Ten-day-old seedling. B,Close-up view of cotyledons showing GUS expres-sion in stipules (s) and hydathodes (hy). C, Detail ofthe stipules. D, Detail of GUS expression at the baseof trichomes (t). E, GUS expression in rosette leafhydathodes. F, Close-up view of a hydathode. G,Flower. H, Embryo. I, Pollen. Photographs are repre-sentative of at least 10 independent experiments.Scale bars 5 500 mm (A, B, E, and G); 50 mm (C, D, F,H, and I).





wounded zone, whereas no GUS activity wasobserved in the middle of the unwounded leaf (Fig.7A). Flavonoid phytoalexins have frequently been pro-posed to participate in plant response to pathogenattack and the corresponding enzymes involved areinduced by pathogens (Bell et al., 1984; Lawton andLamb, 1987; Garcıa-Ponce and Rocha-Sosa, 2000). Weinfected PvACCaseTGUS plants with P. syringae pvtomato/AvrRpm1 (Pst), a pathogen known to establishan incompatible interaction with Arabidopsis. There isstrong PvACCaseTGUS induction by the pathogen,whereas the control mock-treated leaves showed aweak response, probably resulting from manipulation(Fig. 7B). This result is consistent with the role offlavonoids in plant defense and indicated that the2.7-kb promoter fragment is sufficient for this response.

Previously, we showed that JA and ethylene inducePvACCase mRNA accumulation in common bean(Garcıa-Ponce and Rocha-Sosa, 2000; Fig. 1B); there-fore, we decided to analyze PvACCase gene promoterresponsiveness to these stimuli. Seedlings were treatedwith MeJA or the ethylene biosynthesis precursor,ACC, and tested for GUS activity. After both treatments,there was a significant increase in PvACCaseTGUSexpression compared to the untreated control (Fig. 8).

Flavonoids are also scavengers of ROS, which are in-termediary of the defense responses in plant-pathogeninteractions (Yamasaki et al., 1997; Dat et al., 2000);therefore, PvACCase participation in oxidative stresswas also assayed. When PvACCaseTGUS seedlingswere treated with hydrogen peroxide (H2O2), strongerpromoter expression was detected in leaves (Fig. 8).

PvACCase::GUS Expression in HormonePerception Mutants

To elucidate cross talk between different defensesignaling pathways, we generated crosses between JA

(coi1-1) or ethylene (etr1-1) perception mutants andour PvACCaseTGUS transgenic line. The coi1-1 mu-tation is recessive and leads to male sterility andinsensitivity to coronatine and MeJA (Feys et al.,1994). We used several lines that were homozygousfor kanamycin resistance (selection marker to thePvACCase gene promoterTGUS cassette) and segre-gated 3:1 for sensitivity/insensitivity to MeJAwhen grown on Murashige and Skoog medium con-taining MeJA. Accordingly, 25% of the seedlingsshould be homozygous for coi1-1 and homozygousfor PvACCaseTGUS. The etr1-1 mutation is dominantdue to a mutation within the ethylene-binding site andshows ethylene insensitivity and Glc hypersensitivity(Chang et al., 1993; Zhou et al., 1998). Homozygouslines from both crosses were used for analysis.

Exogenous MeJA induced GUS expression in theetr1-1 background, but, as expected, GUS expres-sion was undetectable in the coi1-1 background as aresult of JA insensitivity (Fig. 8). On the other hand,PvACCaseTGUS was induced when ACC was ap-plied to the coi1-1 mutant, but no GUS activity was de-tected in the etr1-1 background (Fig. 8). Taken together,our results suggest that PvACCaseTGUS expressionwas activated through both ethylene and JA signalingpathways.

Pathogen attack and wounding both induced sub-stantial GUS expression in the coi1-1 and etr1-1 mutantbackgrounds. As in the PvACCaseTGUS wild-typeline, wounding caused local staining surrounding thewound site (Fig. 7, A and B). These results indicate thatJA and ethylene signaling pathways can each actindependently to regulate PvACCase induction inresponse to these stimuli or that an entirely differentsignaling pathway is used. To distinguish experimen-tally between these two hypotheses, a double mutant,coi1-1/etr1-1, bearing the PvACCaseTGUS gene pro-moter was generated and challenged as describedabove. GUS expression was absent after pathogen in-fection, MeJA, or ACC treatments (Figs. 7B and 8). Theseobservations confirm the hypothesis that PvACCaseTGUS activation can be mediated by at least two inde-pendent signaling pathways. Surprisingly, we de-tected the same expression pattern during the woundresponse in coi1-1/etr1-1 plants as in wild-type plants,indicating the participation of at least one signaling

Figure 4. Root expression pattern of PvACCase gene promoter. A, GUSstaining in a 3-d-old root. B, GUS staining in the root tip and elongationzone of a 5-d-old root. C, GUS staining in the elongation zone of a 7-d-old root. D, Five-day-old seedling showing GUS activity in the hypocotyl-root transition zone (TZ). E, GUS expression in a lateral root primordium(LRP) of a 7-d-old seedling. F, Close-up view of a developing lateral root(LR) of a 7-d-old seedling. G, Lateral roots of a 14-d-old seedling.Photographs are representative of at least 10 independent experiments.Scale bars 5 25 mm (A–C); 50 mm (D–F); 500 mm (G).

Figure 5. PvACCase mRNA accumulation in organs of common bean.Total RNA (30 mg/lane) of several organs of bean plants was used forRNA-blot analysis. R, Root; S, stem; YL, young leaf of 10-d-old plants;ML, mature leaf of 23-d-old plants; SL, senescent leaf of 57-d-oldplants; FB, floral bud; F, flower. rRNA staining with ethidium bromide isshown as a loading control. A representative experiment of at leastthree independent experiments is shown.





pathway independent of both JA and ethylene(Fig. 7A).

Treatment with IAA induced GUS expression in thecoi1-1 mutant to a level similar to that observed inwild-type transgenic plants. In etr1-1 plants, however,IAA treatment did not induce GUS expression (Fig. 8).This result suggests that IAA activation of PvACCaseexpression most likely reflects ethylene accumulationinduced by auxins because this auxin response isblocked in the etr1-1 background. As expected, in thecoi1-1/etr1-1 double mutant, IAA was unable to inducePvACCaseTGUS expression (Fig. 8).

The impact of H2O2 on PvACCaseTGUS expressionwas also examined in coi1-1 and etr1-1 plants. Asshown in Figure 8, GUS expression was induced byH2O2 in both mutants. In the coi1-1/etr1-1 doublemutant, GUS activity was not detected after H2O2treatment (Fig. 8). These results indicate that H2O2induction of the PvACCase promoter can utilize ei-ther the ethylene- or JA-responsive signaling path-ways, but not the hormone-independent pathway. Theinduction of marker genes responsive to differentstresses and chemicals was examined by reverse tran-scription-PCR analysis as treatment controls. We se-lected the following genes for expression analysis:the JA- and ethylene-induced marker gene PDF1.2(Penninckx et al., 1998), which corresponds to a plantdefensin; the auxin-responsive gene IAA19 (Godaet al., 2004); the oxidative stress-responsive geneGST6 (Wagner et al., 2002); and the ethylene-induciblegene Hel1 (Potter et al., 1993), encoding a hevein-likeprotein with antifungal activity (Supplemental Fig.

S2). Collectively, the treatment regimes indicate thatthere are at least three independent routes by whichbiotic and abiotic challenges can activate PvACCasegene expression (Fig. 9).

DISCUSSION

In plant cells, the cytosolic pool of malonyl-CoAgenerated by ACCase is required to support the bio-synthesis of many secondary phytochemicals impor-tant for plant development, growth, and protection.These phytochemicals include VLCFA, flavonoids,and stilbenes (Ebel and Hahlbrock, 1977; Schroderet al., 1988; Roesler et al., 1994). Although the role ofACCase in flavonoid biosynthesis is well established(Ebel and Hahlbrock, 1977), little is known about theendogenous factors regulating its expression or itstissue and organ distribution in bean or Arabidopsis.We have previously demonstrated that, in bean cellcultures and leaves, PvACCase mRNA accumulates inresponse to elicitor or pathogen infection. Pathogen-induced PvACCase protein and mRNA accumulation

Figure 7. Stress effects on PvACCase gene promoter activity. GUSexpression in 5- to 6-week-old rosette leaves of Col-0, coi1-1, etr1-1,and coi1-1/etr1-1 plants that express the PvACCaseTGUS gene 6 hafter wounding with forceps (A) and 6 h after spraying with 10 mM

MgCl2 as a control and 6 h after spraying with P. syringae pv tomato/AvrRPM1 (B). Figures are representative of at least five independentexperiments. Scale bar 5 1 mm.

Figure 6. Whole-mount mRNA expression patterns in common beanand Arabidopsis roots. A, Whole-mount 3-d-old roots of common beanwere hybridized with sense and antisense PvACCase probes (top).Whole-mount common bean lateral roots hybridized with sense andantisense PvACCase-specific probes (bottom). B, Whole-mount 3-d-oldroots of Arabidopsis hybridized with a sense and antisense AtACC1-specific probe as indicated. A representative experiment of at leastthree independent experiments is shown. Scale bars 5 250 mm (A);25 mm (B).





was reduced by the application of inhibitors of oxy-lipin biosynthesis, suggesting a role for oxylipins asmediators in the induction of the PvACCase gene inresponse to pathogen infection (Garcıa-Ponce andRocha-Sosa, 2000). Similar experiments with inhibitorsof ethylene action indicated that ethylene was alsorequired for pathogen-induced PvACCase mRNA ac-cumulation (Garcıa-Ponce, 2000). To gain further un-derstanding of PvACCase gene regulation in responseto stress and to clarify the roles of JA and ethylene asresponse mediators, PvACCaseTGUS expression wasanalyzed in Arabidopsis transgenic wild-type and inJA or ethylene perception mutant plants; this approachcircumvents the use of inhibitors and thus avoidssecondary inhibitor effects.

The PvACCase control region contains several cis-elements that have been proven to control responses towounding, pathogen infection, and hormones (Fig. 2).These elements are conserved in soybean and Arabi-dopsis cytosolic ACCase genes. Among the motifs area G-box and an H-box separated by 38 bp. Promotersof genes in the phenylpropanoid pathway containthese regulatory elements and, in this context, they arenecessary for flower- and root-specific expression(Faktor et al., 1997). Interestingly, we observed thatPvACCase promoter induction was abolished in con-structs that do not contain the G-box and H-box motifs(data not shown). In addition, adjacent G- and H-boxesin the promoter of the common bean chs15 gene arebound by transcription factor G/HBF-1. This protein isphosphorylated upon elicitor treatment (Droge-Laseret al., 1997). The presence of these motifs in thePvACCase promoter region suggests coordinated reg-ulation with other genes for phenylpropanoid synthe-

sis. A W1-box was also found at 2288 bp upstreamto the ATG start codon in the PvACCase gene pro-moter. The W-box motif has been previously shownto bind WRKY transcription factors (Eulgem et al.,1999). WRKY factors play a key role in regulating the

Figure 8. Effect of hormones and oxidative stresson PvACCase expression. GUS expression in 8-d-old seedlings of Col-0, coi1-1, etr1-1, and coi1-1/etr1-1 plants that express the PvACCaseTGUSgene. Untreated (C) or 6 h after treatment with100 mM MeJA, 200 mM ACC, 1 mM IAA, or 1 mM

H2O2 as indicated above each photograph. Thefigures are representative of at least five indepen-dent experiments. Scale bar 5 0.5 mm.

Figure 9. Scheme of the activation of the PvACCase gene in response tobiotic and abiotic stress. Pathogen- or ROS-induced PvACCase genepromoter activation can utilize either the JA (COI1)- or the ethylene(ETR1)-responsive signaling pathways. In the model, we position ROSdownstream of the pathogen in agreement with the knowledge thatROS are generated during the hypersensitive response caused by anincompatible plant-pathogen interaction (Dat et al., 2000). Flavonoidsproduced in response to pathogens act as ROS scavengers, limiting thedamage caused by this stress condition. IAA-induced PvACCase genepromoter activation is mediated through the capacity of auxins topromote ethylene synthesis. Wounding induces PvACCase gene acti-vation by a signaling pathway independent of both JA and ethylene.





pathogen-induced defense program (Yang et al., 1999)and they also seem to be involved in other specificprocesses, such as trichome development and second-ary metabolite biosynthesis (Walker et al., 1999).

Mutants in the ACC1 gene encoding the cytosolicACCase in Arabidopsis are lethal in embryo develop-ment. Because all known mutants in flavonoid syn-thesis have normal embryos, the absence of VLCFAand their derivatives must be the cause of embryoarrest in acc1 plants (Baud et al., 2003). Interestingly,we detected PvACCase gene promoter-directed GUSactivity in the Arabidopsis embryo (Fig. 3G), suggest-ing a key role of the PvACCase in common beanembryo development too.

Flavonoids are present in growing and maturingtissues of Arabidopsis, including siliques, inflores-cence stems, flowers, stigma, pollen, and cauline androsette leaves (Peer et al., 2001). The heterologous beanpromoter programmed PvACCaseTGUS expressionin the same tissues. To our knowledge, our work is thefirst to report the expression of a cytosolic ACCasegene in the hydathodes, stipules, and at the base ofsome trichomes of cauline and rosette leaves (Fig. 3).Hydathodes are connected to the vascular tissue andthese glands lose water at the leaf tip and in the lobes.These glands are also sites of pathogen entry: Thenecrotrophic bacteria Xanthomonas campestris pv cam-pestris invades Arabidopsis primarily through thehydathodes. Given the relatively fast induction of thesuperoxide dismutase gene in bacteria in the hyda-thodes, the existence of hydathode-localized defensereactions has been suggested (Hugouvieux et al.,1998). Indeed, induced expression of defense-relatedgenes, such as ChS, has been reported (Aloni et al.,2003). Because hydathodes lack structural barriersagainst pathogen entry, expression of the PvACCaseTGUS gene fusion in these regions may indicate thatgene products (i.e. flavonoids) contribute to defense,probably by acting as phytoalexins or scavengers ofROS.

The PvACCase promoter was also active in stipulesand some trichomes (Fig. 3). Hydathodes and stipulesare the primary sites of free IAA production in a leafblade and trichomes are secondary sites (Aloni et al.,2003). In roots, we detected GUS expression in the roottip, elongation zone, lateral root primordia, and thehypocotyl-root transition zone (Fig. 4). Amazingly,the same root regions with GUS activity accumulateboth flavonoids and auxins (Murphy and Taiz, 1999;Murphy et al., 2000; Peer et al., 2001; Ljung et al., 2005).In this context, it should be stressed that, in Arabi-dopsis roots, ChS and chalcone isomerase (Saslowskyand Winkel-Shirley, 2001) are located in sites wherethe PvACCase gene promoter is active (Fig. 4) andPvACCase and ACC1 mRNAs are present (Fig. 6).It has been suggested that flavonoids act as auto-crine effectors that retain auxins in the cells in whichthey are synthesized (or accumulated), altering PIN-FORMED expression and localization and, conse-quently, polar auxin transport (Peer et al., 2004). To

test the possibility that auxin regulates PvACCasegene expression, plants expressing the PvACCaseTGUS fusion were treated with IAA, resulting in GUSactivity in leaf tissues (Fig. 8). In etr1-1 plants, no IAAinduction was observed (Fig. 8), suggesting that IAAaction is mediated through its capacity to induceethylene synthesis (McKeon et al., 1995). Consequently,it is possible that the induction of the PvACCasepromoter responds to a developmental signal andnot directly to IAA.

PvACCase is induced by pathogen infection incommon bean; a full induction requires oxylipinsand ethylene (Garcıa-Ponce, 2000; Garcıa-Ponce andRocha-Sosa, 2000). Now, we first demonstrate thatPvACCase mRNA accumulates after wounding andethylene treatment, reaching a peak at 3 and 6 h,respectively (Fig. 1). Histochemical analysis of trans-genic plants expressing the PvACCaseTGUS fusiongene showed that the PvACCase promoter is inducedupon wounding or pathogen infection of Arabidopsis(Fig. 7, A and B); incubation of seedlings with MeJA orACC also resulted in the induction of GUS activity(Fig. 8). Collectively, these results implicate both hor-mones mediate wounding and pathogen responses. Todeterminate the number and properties of the signaltransduction pathways, we analyzed GUS expressionin two signal perception mutants. Wounded plants inboth the coi1-1 and etr1-1 background showed GUSstaining, as did the coi1-1/etr1-1 double mutants (Fig.7A). Therefore, one signaling pathway is independentof both JA and ethylene. This notion is consistent withrecent studies showing that OPDA induces severalgenes via a COI1-independent signaling pathwayduring the wound response (Taki et al., 2005).

In contrast to wounding, the PvACCase gene pro-moter was activated by Pst infection in wild-type andcoi1-1 or etr1-1 plants; however, in the double mutant,Pst was unable to activate this promoter (Fig. 7B);therefore, JA and ethylene act independently to pro-gram the response of PvACCase to pathogens.

ROS are produced quickly and have an importantrole in triggering responses of plants to biotic andabiotic stresses. Flavonoids are scavengers of ROS inconjunction with peroxidase and are essential for thesurvival of uninfected tissue. The PvACCase genepromoter was activated in response to H2O2 in wild-type and coi1-1 or etr1-1 plants; in the double mutant,however, this promoter was inactive (Fig. 8). In linewith this, it is possible that, in pathogen infectionresponse, ROS act upstream of JA and ethylene forinduction of PvACCase and that either COI1 or ETR1signaling pathways are necessary for this activation.

Results described here provide data on PvACCasetissue expression during normal plant development insites of flavonoid and auxin accumulation. In conclu-sion, PvACCase promoter activity was also inducedunder various stress conditions where metabolic alter-ations were triggered. We propose that the PvACCasegene is regulated by at least three independent routesin biotic and abiotic stress response (Fig. 9).





MATERIALS AND METHODS

Plant Material

Common bean (Phaseolus vulgaris L cv Negro Jamapa) was grown etiolated

in trays with wet paper towels for 10 d. These plants were used for genomic

DNA extraction and library construction. Arabidopsis (Arabidopsis thaliana

ecotype Col-0) was the genetic background for all mutant and transgenic plants

used in this work. The coi1-1 mutants were kindly provided by John G. Turner

(University of East Anglia, UK), whereas the etr1-1 mutant was obtained from

the Arabidopsis Biological Resource Center at Ohio State University.

RNA-Blot Analysis

RNA was prepared as described by Logemann et al. (1987). RNA-blot

hybridization analysis was performed with 30 mg of total RNA per lane.

Hybridization was done in 0.3 M sodium phosphate, pH 7.2, 7% SDS, and 1 mM

EDTA at 65�C with the gene-specific probe for 3#-untranslated region of the

PvACCase gene (AF007803). Blots were washed at low stringency with 1 M

sodium phosphate, pH 7.2, 0.67% SDS.

Promoter Cloning

Genomic libraries were prepared from bean using the Universal Genome

Walker kit (CLONTECH). DNA was digested with seven blunt-ended en-

zymes. The genomic fragments were then ligated to specific adapters pro-

vided in the kit, resulting in seven libraries. Based on the common bean

ACCase gene sequence (GenBank accession no. DQ355997), two gene-specific

primers were designed: GSP1, 5#-GTCTCATTGGCCCAGCTCCTAACAC-

TACGT-3# and GSP2, 5#-GAACTTGACTGCTGCCATTCCATTGTTTGC-3#.

These primers facilitated amplification of the PvACCase 5# regions as detailed

in the manual (CLONTECH). Four fragments resulting from PCR reactions

varied in length in the 5# promoter region, but were identical in the regions of

overlap and were sequenced; 5# sequences upstream of the PvACCase gene

were analyzed for potential regulatory motifs and promoter-like elements.

The HindACC primer was designed to amplify the promoter region and to

create a HindIII restriction site at the 5# end of the amplified fragment (the

HindIII site is indicated by the underlined bases): 5#-TTGAAGCTTTGAA-

AAACATGACACTTGT-3#. Another primer, BamACC, was designed to create

a BamHI site at the 3# end of the amplified fragment (the BamHI site is in-

dicated by the underlined bases): 5#-TACATAGGAGACATACAGAAG-

GATCCCAAT-3#. These primers were used to amplify the promoter region.

The resulting fragment was digested with HindIII and BamHI, column

purified, and cloned into the respective sites in the pBin121 (CLONTECH)

plant transformation vector. The resulting plasmid (ACCase-pBin121) was

used to transform Arabidopsis.

Plant Transformation and Crossing

Arabidopsis was transformed using the floral-dip method (Clough and

Bent, 1998) and subsequently grown to obtain seeds. T2 seeds from transgenic

lines exhibiting 3:1 segregation for kanamycin resistance were selected and

T3-independent homozygous lines exhibiting 100% kanamycin resistance

were recovered. Two independent homozygous transgenic lines (PvACCase::GUS) were crossed with mutant lines coi1-1 and etr1-1. F1 progeny were

allowed to self pollinate and F2 seeds were harvested. F3 seeds from the cross

PvACCaseTGUS/coi1-1 (coi1-1) were maintained heterozygous for coi1-1 be-

cause homozygotes are male sterile. Homozygous coi1-1 F3 plants were

obtained from double selection with 50 mM JA and 50 mg/mL kanamycin.

Homozygous PvACCaseTGUS/etr1-1 (etr1-1) F4 plants were obtained from

double selection with 4% Glc and 50 mg/mL kanamycin. The coi1-1/etr1-1

double-mutant line was constructed by crossing between coi1-1 and etr1-1

mutant lines carrying PvACCaseTGUS. The double mutants were selected on

the basis of their insensitivity to MeJA and resistance to ethylene. Derived

homozygous lines from these crosses were used for all treatments.

Histochemical GUS Assays

GUS activity was detected by histochemical staining following the proce-

dure of Jefferson (1987). Fresh and treated tissues were immersed in GUS

staining buffer (100 mM sodium phosphate, pH 7.0, 10 mM EDTA, 0.5 mM

Triton X-100, 0.5 mM potassium ferrocyanide, 0.5 mM potassium ferricyanide,

2 mM 5-bromo-4-chloro-3-indolyl-b-D-GlcUA substrate predissolved in 0.5%

N-dimethylformamide), and incubated for approximately 16 h at 37�C. The

staining solution was removed and the samples were cleared in 80% ethanol.

Roots were stained and cleared as described by Malamy and Benfey (1997).

Whole-Mount in Situ Hybridization

AtACC1 (At1g36160) and PvACCase (AF007803) sense and antisense

probes corresponding to the coding region and the 3#-untranslated region,

respectively, were in vitro synthesized and digoxigenin labeled (Roche Diag-

nostics). Short riboprobes (an average length of 150 bp) were produced by

alkaline hydrolysis of longer transcripts. Whole-mount in situ hybridization

was performed as described by Friml et al. (2003). Roots were observed using

an Eclipse E600 stereoscopic microscope and an optic microscope equipped

with a digital camera E995 (Nikon).

Plant Treatments

Leaves of 13-d-old bean plants were wounded perpendicularly on the

central vein with dialysis closure clips and incubated for the indicated times

leaving it on. Plants to be treated with ethylene were grown in a standard

hydroponic system for 23 d and incubated with ethephon for the indicated

times. Ethephon was dissolved in 0.5 M sodium phosphate, pH 7.2, and used

at a final 100 mM concentration. Immediately after harvest, all plant material

was immersed in liquid nitrogen prior to RNA extraction.

To improve the visualization of the effect of mechanical wounding, the

middle of rosette leaves of 5- to 6-week-old Arabidopsis plants was crushed

once or twice, with a dissection forceps and incubated for 6 h. Infection with

Pseudomonas syringae was performed by spraying bacteria on the adaxial side

of rosette leaves of 5- to 6-week-old plants using suspension of the strain

DC300/avr RPM1 at 1 3 108 cfu/mL in 10 mM MgCl2, 0.02% (v/v) Silwet L-77.

Treated plants were incubated for 6 h in an airtight transparent plastic box in a

lighted growth chamber. For chemical treatments, seedlings were grown over

sterilized nylon filters in a vertical orientation for 8 d and then transferred

from standard Murashige and Skoog medium to fresh Murashige and Skoog

medium containing standard concentrations reported for gene expression

analysis. The concentrations used were 100 mM MeJA (Onate-Sanchez and

Singh, 2002); 200 mM ACC (He et al., 2004); 1 mM H2O2 (Wagner et al., 2002);

and 1 mM IAA (Goda et al., 2004). After chemical treatment, plants were

incubated for 6 h in a growth chamber. With this procedure, we avoided

wounding the seedling during transfer. Control seedlings were incubated

under the same conditions without any treatment.

Sequence data have been deposited in the DDBJ/EMBL/GenBank nucle-

otide sequence databases under accession number DQ355997.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Effect of auxin application on PvACCase::GUS

expression in roots.

Supplemental Figure S2. RT-PCR analysis of marker gene expression.

ACKNOWLEDGMENTS

We thank Dr. Virginia Walbot, Dr. Gladys Cassab, Dr. Helena Porta, Dr.

Arturo Guevara, and Dr. Jose A. Rocha for critical reading and helpful

comments on the manuscript.

Received June 23, 2006; accepted August 18, 2006; published August 25, 2006.

LITERATURE CITED

Aloni R, Schwalm K, Langhans M, Ulrich CI (2003) Gradual shifts in sites

of free-auxin production during leaf-primordium development and

their role in vascular differentiation and leaf morphogenesis in Arabi-

dopsis. Planta 216: 841–853

Baud S, Bellec Y, Miquel M, Bellini C, Caboche M, Lepiniec L, Faure JD,

Rochat C (2004) gurke and pasticcino3 mutants affected in embryo





development are impaired in acetyl-CoA carboxylase. EMBO Rep 5:

515–520

Baud S, Guyon V, Kronenberger J, Wuilleme S, Miquel M, Caboche M,

Lepiniec L, Rochat C (2003) Multifunctional acetyl-CoA carboxylase I is

essential for very long chain fatty acid elongation and embryo devel-

opment in Arabidopsis. Plant J 33: 75–86

Bell JN, Dixon RA, Bailey JA, Rowell PM, Lamb CJ (1984) Differential

induction of chalcone synthase mRNA activity at the onset of phyto-

alexin accumulation in compatible and incompatible plant-pathogen

interactions. Proc Natl Acad Sci USA 81: 3384–3388

Boerjan W, Cervera MT, Delarue M, Beeckman T, Dewitte W, Bellini C,

Caboche M, Van Onckelen H, Van Montagu M, Inze D (1995) Super-

root, a recessive mutation in Arabidopsis, confers auxin overproduc-

tion. Plant Cell 7: 1405–1419

Brown DE, Rashotte AM, Murphy AS, Normanly J, Tague BW, Peer WA,

Taiz L, Muday GK (2001) Flavonoids act as negative regulators of auxin

transport in vivo in Arabidopsis. Plant Physiol 126: 524–535

Buer CS, Sukumar P, Muday GK (2006) Ethylene modulates flavonoid

accumulation and gravitropic responses in roots of Arabidopsis thaliana.

Plant Physiol 140: 1384–1396

Casimiro I, Marchant A, Bhalerao RP, Beekma T, Dhooge S, Swarup R,

Graham N, Inze D, Sandberg G, Casero PJ, et al (2001) Auxin transport

promotes Arabidopsis lateral root initiation. Plant Cell 13: 843–852

Chang C, Kwok SF, Bleecker AB, Meyerowitz EM (1993) Arabidopsis

ethylene response gene ETR1: similarity of product to 2-component

regulators. Science 262: 539–544

Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agro-

bacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:

735–743

Dat J, Vandenabeele S, Vranova E, Van Montagu M, Inze D, Van

Breusegem F (2000) Dual action of the active oxygen species during

plant stress responses. Cell Mol Life Sci 57: 779–795

Devoto A, Ellis C, Magusin A, Chang H-S, Chilcott C, Zhu T, Turner JG

(2005) Expression profiling reveals COI1 to be a key regulator of genes

involved in wound- and methyl jasmonate-induced secondary metab-

olism, defence, and hormone interactions. Plant Mol Biol 58: 497–513

Dittrich H, Kutchan TM, Zenk MH (1992) The jasmonate precursor

12-oxo-phytodienoic acid induces phytoalexin synthesis in Petroselinum

crispum cell cultures. FEBS Lett 309: 33–36

Dixon RA, Pavia NL (1995) Stress-induced phenylpropanoid metabolism.

Plant Cell 7: 1085–1097

Droge-Laser W, Kaiser A, Lindsay WP, Halkier BA, Loake GJ, Doerner P,

Dixon RA, Lamb C (1997) Rapid stimulation of a soybean protein-serine

kinase that phosphorylates a novel bZIP DNA-binding protein, G/HBF-1,

during the induction of early transcription-dependent defenses. EMBO J

16: 726–738

Ebel J, Hahlbrock K (1977) Enzymes of flavone and flavonol-glycoside

biosynthesis: coordinated and selective induction in cell-suspension

cultures of Petroselinum hortense. Eur J Biochem 75: 201–209

Ecker JR, Davis RW (1987) Plant defense genes are regulated by ethylene.

Proc Natl Acad Sci USA 84: 5202–5206

Eulgem T, Rushton PJ, Schmelzer E, Hahlbrook K, Somssich IE (1999)

Early nuclear events in plant defense: rapid gene activation by WRKY

transcription factors. EMBO J 18: 4689–4699

Evans ML, Ishikawa H, Estelle MA (1994) Responses of Arabidopsis roots

to auxin studied with high temporal resolution: comparison of wild type

and auxin response mutants. Planta 194: 215–222

Faktor O, Loake G, Dixon RA, Lamb CJ (1997) The G-box and H-box in a

39 bp region of a French bean chalcone synthase promoter constitute a

tissue-specific regulatory element. Plant J 11: 1105–1113

Feys BJF, Benedetti CE, Penfold CN, Turner JG (1994) Arabidopsis

mutants selected for resistance to the phytotoxin coronatine are male-

sterile, insensitive to methyl jasmonate, and resistant to a bacterial

pathogen. Plant Cell 6: 751–759

Franceschi VR, Grimes HD (1991) Induction of soybean vegetative storage

proteins and anthocyanins by low-level atmospheric methyl jasmonate.

Proc Natl Acad Sci USA 88: 6745–6749

Friml J, Benkova E, Mayer U, Palme K, Muster G (2003) Automated whole

mount localisation techniques for plant seedlings. Plant J 34: 115–124

Garcıa-Ponce B (2000) Caracterizacion molecular de la respuesta a estres

de la acetil CoA carboxilasa de frijol (Phaseolus vulgaris L.). PhD thesis.

Instituto de Biotecnologıa-Universidad Nacional Autonoma de Mexico,

Cuernavaca, Morelos, Mexico

Garcıa-Ponce B, Rocha-Sosa M (2000) The octadecanoid pathway is

required for pathogen-induced multifunctional acetyl-CoA carboxylase

accumulation in common bean (Phaseolus vulgaris L.). Plant Sci 157:

181–190

Goda H, Sawa S, Asami T, Fujioka S, Shimada Y, Yoshida S (2004)

Comprehensive comparison of auxin-regulated and brassinosteroid-

regulated genes in Arabidopsis. Plant Physiol 134: 1555–1573

Harwood JL (1988) Fatty acid metabolism. Annu Rev Plant Physiol Plant

Mol Biol 39: 101–138

He X, Zhang Z, Yan D, Zhang J, Chen S (2004) A salt responsive receptor-

like kinase gene regulated by the ethylene signaling pathway encodes a

plasma membrane serine/threonine kinase. Theor Appl Genet 109:

377–383

Hugouvieux V, Barber CE, Daniels MJ (1998) Entry of Xanthomonas

campestris pv campestris into hydathodes of Arabidopsis thaliana leaves:

a system for studying early infection events in bacterial pathogenesis.

Mol Plant Microbe Interact 11: 537–543

Jefferson RA (1987) Assaying chimeric genes in plants: the GUS gene

fusion system. Plant Mol Biol Rep 5: 387–405

Kajiwara T, Furutani M, Hibara K, Tasaka M (2004) The GURKE gene

encoding an acetyl-CoA carboxylase is required for partitioning the

embryo apex into three subregions in Arabidopsis. Plant Cell Physiol 45:

1122–1128

Konishi T, Sasaki Y (1994) Compartmentation of two forms of acetyl-CoA

carboxylase in plants and the origin of their tolerance towards herbi-

cides. Proc Natl Acad Sci USA 91: 3598–3601

Konishi T, Shinohara K, Yamada K, Sasaki Y (1996) Acetyl CoA carbox-

ylase in higher plants: most plants other than gramineae have both the

prokaryotic and the eukaryotic forms of this enzyme. Plant Cell Physiol

37: 117–122

Landry LG, Chapple CCS, Last RL (1995) Arabidopsis mutants lacking

phenolic sunscreens exhibit enhanced ultraviolet-B injury and oxidative

damage. Plant Physiol 109: 1159–1166

Lawton MA, Lamb CJ (1987) Transcriptional activation of plant defense

genes by fungal elicitor, wounding and infection. Mol Cell Biol 7:

335–341

Lescot M, Dehais P, Thijs G, Marchal K, Moreau Y, Van de Peer Y, Rouze

P, Rombauts S (2002) PlantCARE, a database of plant cis-acting regu-

latory elements and a portal to tools for in silico analysis of promoter

sequences. Nucleic Acids Res 30: 325–327

Liu Y, Hoffman NE, Yang SF (1983) Relationship between the malonation

of 1-aminocyclopropane-1-carboxylic acid and D-amino acids in mung-

bean hypocotyls. Planta 158: 437–441

Ljung K, Hull AK, Celenza J, Yamada M, Estelle M, Normanly J,

Sandberg G (2005) Sites and regulation of auxin biosynthesis in

Arabidopsis roots. Plant Cell 17: 1090–1104

Logemann J, Schell J, Willmitzer L (1987) Improved method for the

isolation of RNA from plant tissues. Anal Biochem 163: 16–20

Lois R, Buchanan BB (1994) Severe sensitivity to ultraviolet radiation in an

Arabidopsis mutant deficient in flavonoid accumulation. Planta 194:

504–509

Malamy JE, Benfey PN (1997) Organization and cell differentiation in

lateral roots of Arabidopsis thaliana. Development 124: 33–44

McKeon T, Fernandez-Maculet JC, Yang SF (1995) Biosynthesis and

metabolism of ethylene. In PJ Davis, ed, Plant Hormones and Their

Role in Plant Growth and Development. Kluwer Academic Publishers,

Dordrecht, The Netherlands, pp 118–139

Murphy A, Peer WA, Taiz L (2000) Regulation of auxin transport by

aminopeptidases and endogenous flavonoids. Planta 211: 315–324

Murphy AS, Taiz L (1999) Naphthyphthalamic acid is enzymatically

hydrolyzed at the hypocotyl-root transition zone and other tissues of

Arabidopsis thaliana seedlings. Plant Physiol Biochem 37: 413–430

O’Donnell PJ, Calvert C, Atzorn R, Wasternack C, Leyser HMO, Bowles

DJ (1996) Ethylene as a signal mediating the wound response of tomato

plants. Science 274: 1914–1917

Onate-Sanchez L, Singh KB (2002) Identification of Arabidopsis ethylene-

responsive element binding factors with distinct induction kinetics after

pathogen infection. Plant Physiol 128: 1313–1322

Parchmann S, Gundlanch H, Mueller MJ (1997) Induction of 12-oxo-

phytodienoic acid in wounded plants and elicited plant cell cultures.

Plant Physiol 115: 1057–1064

Peer WA, Bandyopadhyay A, Blakeslee JJ, Makam SN, Chen RJ, Masson PH,

Murphy AS (2004) Variation in expression and protein localization of the





PIN family of auxin efflux facilitator proteins in flavonoid mutants with

altered auxin transport in Arabidopsis thaliana. Plant Cell 16: 1898–1911

Peer WA, Brown DE, Tague BW, Muday GK, Taiz L, Murphy AS (2001)

Flavonoid accumulation patterns of transparent testa mutants of Arabi-

dopsis. Plant Physiol 126: 536–548

Penninckx IA, Thomma BP, Buchala A, Metraux JP, Broekaert WF (1998)

Concomitant activation of jasmonate and ethylene response pathways is

required for induction of a plant defensin gene in Arabidopsis. Plant

Cell 10: 2103–2113

Potter S, Uknes S, Lawton K, Winter AM, Chandler D, DiMaio J,

Novitzky R, Ward E, Ryals J (1993) Regulation of a Hevein-like gene

in Arabidopsis. Mol Plant Microbe Interact 6: 680–685

Reymond P, Weber H, Damond M, Farmer EE (2000) Differential gene

expression in response to mechanical wounding and insect feeding in

Arabidopsis. Plant Cell 12: 707–720

Roesler KR, Shorrosh BS, Ohlrogge JB (1994) Structure and expression of

an Arabidopsis acetyl-coenzyme A carboxylase gene. Plant Physiol 105:

611–617

Rojo E, Leon J, Sanchez-Serrano JJ (1999) Cross-talk between wound

signaling pathways determines local versus systemic gene expression in

Arabidopsis thaliana. Plant J 20: 135–142

Saslowsky D, Winkel-Shirley B (2001) Localization of flavonoid enzymes

in Arabidopsis roots. Plant J 27: 37–48

Schroder G, Brown JWS, Schroder J (1988) Molecular analysis of revestarol

synthase: cDNA genomic clones and relationship with chalcone syn-

thase. Eur J Biochem 172: 161–169

Schulte W, Topfer R, Stracke R, Schell J, Martini N (1997) Multi-functional

acetyl-CoA carboxylase from Brassica napus is encoded by a multi-gene

family: indication for plastidic localization of at least one isoform. Proc

Natl Acad Sci USA 94: 3465–3470

Stintzi A, Weber H, Reymond P, Browse J, Farmer EE (2001) Plant defense

in the absence of jasmonic acid: the role of cyclopentenones. Proc Natl

Acad Sci USA 98: 12837–12842

Taki N, Sasaki-Sekimoto Y, Obayashi T, Kikuta A, Kobayashi K, Ainai T,

Yagi K, Sakurai N, Suzuki H, Masuda T, et al (2005) 12-Oxo-phyto-

dienoic acid triggers expression of a distinct set of genes and plays a role

in wound-induced gene expression in Arabidopsis. Plant Physiol 139:

1268–1283

Wagner U, Edwards R, Dixon DP, Mauch F (2002) Probing the diversity of

the Arabidopsis glutathione S-transferase gene family. Plant Mol Biol

49: 515–532

Walker AR, Davison PA, Bolognesi-Winfield AC, James CM, Srinivasan

N, Blundell TL, Esch JJ, Marks MD, Gray JC (1999) The TRANSPAR-

ENT TESTA GLABRA 1 locus, which regulates trichome differentiation

and anthocyanin biosynthesis in Arabidopsis, encodes a WD40 repeat

protein. Plant Cell 11: 1337–1350

Yamasaki H, Sakihama Y, Ikehara N (1997) Flavonoid-peroxidase reaction

as a detoxification mechanism of plant cells against H2O2. Plant Physiol

115: 1405–1412

Yang P, Chen Ch, Wang Z, Fan B, Chen Z (1999) A pathogen- and salicylic

acid-induced WRKY DNA-binding activity recognizes the elicitor re-

sponse element of the tobacco class I chitinase gene promoter. Plant J 18:

141–149

Zhou L, Jang JC, Jones TL, Sheen J (1998) Glucose and ethylene signal

transduction crosstalk revealed by an Arabidopsis glucose-insensitive

mutant. Proc Natl Acad Sci USA 95: 10294–10299





hormonal and stress induction of the gene encoding · et al., 1998), but, after wounding, an...

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