Bestatin, an Inhibitor of Aminopeptidases, Provides a Chemical Genetics Approach to Dissect

Bestatin, an Inhibitor of Aminopeptidases, Providesa Chemical Genetics Approach to Dissect JasmonateSignaling in Arabidopsis1[W][OA]

Wenguang Zheng, Qingzhe Zhai, Jiaqiang Sun, Chang-Bao Li, Lei Zhang, Hongmei Li, Xiaoli Zhang,Shuyu Li, Yingxiu Xu, Hongling Jiang, Xiaoyan Wu, and Chuanyou Li*

State Key Laboratory of Plant Genomics and Center for Plant Gene Research, Institute of Geneticsand Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China (W.Z., Q.Z., J.S.,C.-B.L., L.Z., H.L., X.Z., S.L., Y.X., H.J., X.W., C.L.); Graduate School of the Chinese Academyof Sciences, Beijing 100039, China (W.Z., L.Z., H.L., X.Z., S.L., Y.X.); and Horticulture College (Q.Z.)and Key Laboratory of Crop Biology, Agronomy College (C.-B.L., X.W.), Shandong Agricultural University,Taian 271018, China

Bestatin, a potent inhibitor of some aminopeptidases,was shownpreviously to be apowerful inducer ofwound-response genes intomato (Lycopersicon esculentum). Here, we present several lines of evidence showing that bestatin specifically activates jasmonicacid (JA) signaling in plants. First, bestatin specifically activates the expression of JA-inducible genes in tomato and Arabidopsis(Arabidopsis thaliana). Second, the induction of JA-responsive genes by bestatin requires the COI1-dependent JA-signalingpathway, but does not depend strictly on JA biosynthesis. Third, microarray analysis using Arabidopsis whole-genome chipdemonstrates that the gene expression profile of bestatin-treated plants is similar to that of JA-treated plants. Fourth, bestatinpromotes a series of JA-related developmental phenotypes. Taken together, the unique action mode of bestatin in regulating JA-signaled processes leads us to the hypothesis that bestatin exerts its effects through the modulation of some key regulators in JAsignaling.We have employed bestatin as an experimental tool to dissect JA signaling through a chemical genetic screening, whichyielded a collection of Arabidopsis bestatin-resistant (ber) mutants that are insensitive to the inhibitory effects of bestatin on rootelongation. Further characterization efforts demonstrate that some ber mutants are defective in various JA-induced responses,which allowed us to classify the ber mutants into three phenotypic groups: JA-insensitive ber mutants, JA-hypersensitive bermutants, and mutants insensitive to bestatin but showing normal response to JA. Genetic and phenotypic analyses of the bermutants with altered JA responses indicate that we have identified several novel loci involved in JA signaling.

Plants defend themselves against insect attack,wounding, and some pathogen infection by activat-ing the expression of genes involved in herbivoredeterrence, wound healing, and other defense-relatedprocesses. A fascinating feature of induced defenseresponses is their occurrence both locally at the site ofwounding and systemically in undamaged partsthroughout the plant (Green and Ryan, 1972). Wound-inducible proteinase inhibitors (PIs) in tomato (Lyco-persicon esculentum) provide an attractive model

system in which to identify the proposed woundsignals for defense genes expression. Among the iden-tified candidate signals that can activate wound-induced PIs expression in tomato are systemin, an18-amino acid peptide, and jasmonic acid (JA), anoxylipin-derived phytohormone (Farmer and Ryan,1990, 1992; Pearce et al., 1991; Constabel et al., 1995;Bowles, 1998; Walling, 2000; Leon et al., 2001; Browse,2005). A wealth of evidence indicates that systeminand JA actually work together in the same signalingpathway to promote systemic expression of PIs andother defense-related genes (Constabel et al., 1995;Bowles, 1998). However, relatively little is knownabout the specific roles of systemin and JA in long-distance wound signaling. Recently, a series of graftingexperiments conducted with tomato mutants defectivein JA biosynthesis or signaling and, as a consequence,compromised in systemic defense responses led to thecurrent notion that systemin acts at or near the site ofwounding (locally) to amplify the production of JA,which in turn acts as a long-distance wound signal topromote defense genes expression (Li et al., 2002; Ryanand Moura, 2002; Turner et al., 2002; Stratmann, 2003;Howe, 2004; Schilmiller and Howe, 2005). These stud-ies challenge the previous paradigm that systemin is

1 This work was supported by the National Natural ScienceFoundation of China (grant nos. 30425033 and 30530440 to C.L.) andthe Chinese Academy of Sciences (grant no. CXTD–S2005–2 to C.L.).

* Corresponding author; e-mail [email protected]; fax 8610–6487–3428.

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:Chuanyou Li ([email protected]).

[W] The online version of this article contains Web-only data.[OA] Open Access articles can be viewed online without a sub-

scription.Article, publication date, and citation information can be found at

www.plantphysiol.org/cgi/doi/10.1104/pp.106.080390.

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the sole long-distance wound signal and ascribe JA acentral role in regulating systemic defense responsesin virtually all species throughout the plant kingdom.In addition to defense, the jasmonate family of signal-ing molecules, including JA, methyl JA (MeJA), andother bioactive derivatives (collectively referred tohere as jasmonates), also play important roles in regu-lating developmental processes such as root growth,tuberization, senescence, and reproduction (Creelmanand Mullet, 1997; Turner et al., 2002; Wasternack andHause, 2002; Devoto and Turner, 2003; Browse, 2005).In spite of the importance of jasmonates as stressand growth regulators is well recognized, relativelylittle is known about the molecular mechanisms gov-erning JA perception and subsequent signaling eventsleading to activation of downstream JA-responsivegenes.Conventional forward genetic screens for JA-

resistant (insensitive) mutants have led to the identi-fication of a few molecular players in the JA-signalingnetwork, includingubiquitin-mediatedproteolysisma-chinery (Turner et al., 2002; Devoto and Turner, 2003;Browse, 2005). A significant advance in our under-standing of JA signaling came from the analysis of theArabidopsis (Arabidopsis thaliana) coronatine insensitive1 (coi1) mutant that is insensitive to JA. The identifi-cation of COI1 encoding an F-box protein (Xie et al.,1998) and the demonstration that COI1 interacts withSkp1 and Cullin1 to assemble a functional SCFCOI1

ubiquitin ligase complex in vivo (Devoto et al., 2002;Xu et al., 2002) suggest that a ubiquitin-mediatedprotein degradation machinery is involved in JA sig-naling. Significantly, it was demonstrated that loss offunction of a tomato homolog of COI1 (LeCOI1) resultsin a JA-insensitive phenotype both in terms of defenseresponse and development, suggesting the existenceof a COI1 function is conserved among plant speciesother than Arabidopsis (Li et al., 2004). Moreover,plants deficient in the general regulators of SCFcomplexes, such as Cullin1 (Ren et al., 2005), SGT1b(Gray et al., 2003), AXR1 (Leyser et al., 1993), and CSN(Feng et al., 2003), also show impaired JA responses.Taken together, accumulating evidence shows thatthe ubiquitin-mediated protein degradation process,which also has been demonstrated in many differenthormone-signaling pathways, is a central regulatorymechanism in JA signaling.By classic forward genetic screening, other impor-

tant components of the JA-response pathway have alsobeen identified. Compared with coi1, the JA-resistantmutants jar1 (Staswick et al., 1992) and jin1 (Bergeret al., 1996) show a relatively weak phenotype inresponse to JA. JAR1 encodes an enzyme that has JAadenylation activity to form JA-amino acid conjugates,suggesting covalent modification of JA plays a keyrole in its action (Staswick et al., 2002). JIN1 encodesa nuclear-localized bHLH-type transcription factorknown as AtMYC2 (Lorenzo et al., 2004). The tomatohomologs of AtMYC2, named JAMYC2 and JA-MYC10, specifically recognize a T/G box in the pro-

moters of JA-responsive genes and activate theirexpression (Boter et al., 2004). To identify negativeregulators of JA signaling, several groups conductedgenetic screens for constitutive JA-response mutants(Ellis and Turner, 2001; Hilpert et al., 2001; Xu et al.,2001). To date, only one of these genes (CEV1) has beenidentified. The finding that CEV1 encodes a cellulosesynthase suggests a link between cell wall biosynthesisand JA signaling (Ellis et al., 2002).

However, compared to the five classic and otherplant hormones, our knowledge of the JA-signalingpathway is still very limited. For example, extensive con-ventional forward genetic screens failed to identify theJA receptor(s) and the substrate(s) of the SCFCOI1

ubiquitin ligase complex, two of the most critical andapparent gaps in the understanding of JA signal-ing. The inability to identify the JA receptor(s), SCFCOI1

substrates, and other components in forward geneticscreens suggests functional redundancy or lethalmutation in the JA perception and other signalingapparatus. With this in mind, we are looking for analternative approach that may complement the limi-tations of conventional genetics to further dissect JAsignaling. So-called ‘‘chemical genetics’’ is a powerfulapproach to dissect biological processes that may beintractable using conventional genetics because of genefunction redundancy or lethality (Surpin et al., 2005).Chemical genetics is centered around the tenet thatsmall organic molecules can be used like mutations inclassic genetics to modulate protein functions and toassist in delineation of biological pathways (Blackwelland Zhao, 2003). Once small molecules that affect(inhibit or activate) a biological process of interest areidentified, integration of the small molecule-basedforward chemical genetics with the ever-growing ge-nomics tools in model plant system (for example,Arabidopsis) will significantly facilitate the identifica-tion of relevant gene products, including targets of theactive compounds, which are intractable by the classicgenetics approach. Indeed, recent work has demon-strated the feasibility of this approach in studies ofseveral biological pathways, including auxin signal-ing (Blackwell and Zhao, 2003; Zhao et al., 2003;Armstrong et al., 2004; Dai et al., 2005), brassinosteroidsignaling (Asami et al., 2003), vacuole sorting (Zouharet al., 2004), endomembrane trafficking, and gravitrop-ism (Surpin et al., 2005).

To identify new components in JA signaling, wehave developed a forward chemical genetic screen,which combines the advantages of chemical geneticsand the classic genetic screen, to identify novel mu-tants with altered JA responses. Bestatin, a potentinhibitor of some aminopeptidases in plants and an-imals, was shown previously to be a powerful inducerof wound-response genes in tomato (Schaller et al.,1995). Bestatin has been widely used as an experimen-tal tool in elucidating the physiological role of somemammalian exopeptidases in the regulation of theimmune system, in the growth of tumors, and in thedegradation of cellular proteins (Scornik and Botbol,

The Power of Chemical Genetics to Dissect Jasmonate Signaling

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2001). We have conducted a series of genetic and mole-cular analyses in tomato and Arabidopsis, two ofthe most extensively studied model systems for JAresponses, showing that bestatin specifically modu-lates JA signaling. Bestatin specifically activates theexpression of JA-inducible defense-related genes, andthe induction kinetics of these genes in response tobestatin are similar to those in response to JA. Fur-thermore, the action of bestatin to activate defensegene expression requires the function of the F-boxprotein COI1/Jai1, a central regulator of JA-signaling,but does not depend strictly on JA biosynthesis.Microarray analysis using Arabidopsis whole-genomechip demonstrates that the gene transcriptional profileof bestatin-treated plants is similar to that of JA-treatedplants. Finally, bestatin promotes several JA-relateddevelopmental phenotypes. Taken together, the uniqueaction mode of bestatin in regulating JA-signaledprocesses leads us to the hypothesis that bestatinprobably exerts its effects by modulating the JA-signaling pathway. To test this hypothesis and, more-over, to identify novel components involved in JAresponses, we have employed bestatin as an experi-mental tool to dissect JA signaling using a chemicalgenetics approach, which yielded a collection of Arabi-dopsis bestatin-resistant (ber) mutants insensitive to theinhibitory effects of bestatin on primary root elon-gation. Further characterization efforts demonstratethat some ber mutants are defective in various JA- andwound-induced responses, which allowed us to clas-sify them into three phenotypic groups: mutants in-sensitive to both bestatin and JA, mutants insensitiveto bestatin but hypersensitive to JA, and mutantsinsensitive to bestatin but showing normal responseto JA. Among the three groups of mutants, the secondgroup, which is insensitive to bestatin but is hyper-sensitive to JA, is distinct from other identifiedJA-related mutants. Our ongoing effort to identifythe gene functions defined by these novel ber mutantspromises to shed new light on the molecular basis ofJA signaling and on the molecular mechanisms of thebiological function of bestatin in plants.

RESULTS

Bestatin Specifically Activates JA Signaling

in Tomato and Arabidopsis

It was previously reported that bestatin is a power-ful inducer of tomato defense genes, which also areinduced by herbivore attack, mechanical wounding,and the potent wound-response elicitors, such as sys-temin and JA (Schaller et al., 1995). Given that JAworks as a systemic wound signal and plays a centralrole in regulating defense genes expression in re-sponse to herbivore attack and mechanical wounding,we further compared the action mode of bestatin withthat of JA in tomato and Arabidopsis, two of the mostextensively studied systems on JA and wound signal-

ing. The induction kinetics of proteinase inhibitor II(PI-II) mRNA, a well-known representative marker ofa group of wound-inducible mRNAs in tomato, inresponse to exogenous bestatin and JA, was found tobe similar (Fig. 1A). Significant increase in PI-IImRNAlevels was initially observed 2 h after feeding plantswith both bestatin and JA, and mRNA abundance

Figure 1. Activation of JA-inducible genes by bestatin in tomato. A,Induction of PI-IImRNA by bestatin and JA. RNAwas isolated from wild-type (WT) tomato leaves at the times shown after treatment of the cuttingstems with bestatin (40 nmol/plant, dissolved in 15 mM phosphatebuffer), JA (20 nmol/plant, dissolved in 15 mM phosphate buffer), orbuffer control (15 mM phosphate buffer). RNA-blot hybridization wasperformed using 32P-labeled cDNAs for PI-II. A duplicate gel stainedwith ethidium bromide was used as RNA loading control. B, Effects of thespr2 and jai1-1 mutations on bestatin- and JA-induced PI-II mRNAaccumulation. Eighteen-day-old wild-type, spr2, and jai1-1 plants weretreated with bestatin, JA, and buffer control as described in A. Twelvehours after treatment, RNA was isolated from leaves of each genotypeand probed with 32P-labeled PI-II cDNA. A duplicate gel stained withethidium bromide was used as RNA loading control. C, Effects of the spr2and jai1-1 mutations on bestatin- and JA-induced PI-II protein accumu-lation. Eighteen-day-old wild-type, spr2, and jai1-1 plants were treatedwith buffer control (white bars), JA (black bars), and bestatin (gray bars),as described in A. Twenty-four hours after treatment, PI-II protein levelswere measured. Values represent mean 6 SD of 12 plants per genotype.

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continued to rise during the time course, up to 24 h(Fig. 1A).To examine whether the action of bestatin to activate

the expression of JA-related defense genes is ubiqui-tous, we investigated the action of bestatin inArabidopsis. Treatment of wild-type plants with ex-ogenous bestatin readily induced enhanced expres-sion of VEGETATIVE STORAGE PROTEIN1 (AtVSP1;At5g24780), a widely used marker gene of JA re-sponses in Arabidopsis (Fig. 2B). Moreover, the induc-tion kinetics ofAtVSP1 transcript in response to bestatinis very similar to that in response to JA (Fig. 2, A andB). We further constructed a JA reporter line in whichthe promoter of the JA-inducible AtVSP1 gene wasfusedwith the b-glucuronidase (GUS) reporter. Bestatininduces the expression of AtVSP1-GUS throughoutthe plant, with highest expression in young leaves(Fig. 2E). Moreover, the GUS expression pattern of theJA reporter line in response to bestatin is similar to that

in response to JA (Fig. 2, D and E). Taken together,these data demonstrate that bestatin activates JA sig-naling both in tomato and Arabidopsis.

The Action of Bestatin to Activate JA SignalingStrictly Depends on COI1 Function

To determine whether bestatin-induced defensegene expression requires a functional JA-signalingpathway, we further compared the capacity of exoge-nous bestatin and JA to activate JA-inducible markergenes in tomato and Arabidopsis mutants that areknown to be defective in JA biosynthesis or signaling.Application of bestatin to the tomato spr2 mutant,which is deficient in JA biosynthesis (Li et al., 2003),induced the accumulation of PI-II protein to levelscomparable to those in wild-type plant (Fig. 1C).However, bestatin failed to induce PI-II accumulationin the jai1-1 plant (Fig. 1C), which is a loss-of-functionmutant of the tomato homolog of COI1, an F-boxprotein required for JA signaling (Li et al., 2004). Thecapacity of bestatin to induce PI-II protein accumula-tion in wild-type, spr2, and jai1-1 plants is very similarto that of JA, as shown in our parallel experiments (Fig.1C). We also examined the response of spr2 and jai1-1to exogenous bestatin at the level of PI-II transcriptaccumulation. For these experiments, PI-II transcriptlevels were measured 12 h after treatment with bestatinor JA, and the results obtained were in good agree-ment with the measurements of PI-II protein levels.Both bestatin and JA induced the accumulation of PI-IItranscripts in wild-type and spr2 plants, but not injai1-1 plants (Fig. 1B). These results demonstrate thatthe bestatin-induced PI-II accumulation in tomatoleaves requires the Jai1/COI1-dependent JA-signalingpathway, but does not depend on JA biosynthesis.Consistent with the results obtained from the tomatosystem, both bestatin and JA induce AtVSP1 expres-sion in wild-type Arabidopsis plants but not in coi1plants (Fig. 2, A and B), a well-known JA-signalingmutant in Arabidopsis (Feys et al., 1994; Xie et al.,1998). These results indicate that the induction of

Figure 3. Diagrams of the number of overlapping or nonoverlappinggenes up-regulated (A) or down-regulated (B) in response to bestatin(left) and MeJA (right) treatments. Expression data for all of the dif-ferentially regulated genes are listed in Supplemental Tables I to III.

Figure 2. Activation of JA-inducible genes by bestatin in Arabidopsis. Aand B, Parallel experiments to show the induction of the ArabidopsisAtVSP1 expression in wild type and coi1 mutant by JA (A) and bestatin(B). Two-week-old Arabidopsis seedlings (Col-0 wild type and coi1mutant) were treated with 50 mM MeJA, 50 mM bestatin, or not treated(0), and samples were harvested at indicated time points. Total RNAwas isolated and hybridized to 32P-labeled AtVSP1 cDNA, as describedin ‘‘Materials and Methods.’’ A duplicate gel stained with ethidiumbromide was used as RNA loading control. C to E, Activation of the JAreporter gene AtVSP::GUS by bestatin and JA. Seedlings of theArabidopsis AtVSP1::GUS transgenic line grown on MS medium (C),or MS medium containing 25 mM bestatin (D) or 25 mM MeJA (E) for 9 dwere visualized by GUS staining assay.



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AtVSP1 expression by bestatin, just like by JA, alsooccurs through the COI1-dependent JA-signaling path-way in Arabidopsis. Taken together, our results indi-cate that bestatin promotes plant defense responsesby activating the JA-signaling pathway, and furthersuggest that bestatin might exert its action down-stream of JA biosynthesis but upstream of the F-boxprotein COI1.

Gene Transcriptional Profile of Bestatin-TreatedArabidopsis Plants Is Similar to That ofJA-Treated Plants

To gain a deeper understanding on the action mech-anism of bestatin, we performed a microarray analysisusing the Arabidopsis whole-genome chip (Affyme-trix) to compare the global gene transcriptional pro-files regulated by bestatin and MeJA in wild-typeArabidopsis seedlings. The arrays of bestatin- orMeJA-treated samples at the four time points tested(15, 30, 60, and 360 min) were compared with that ofuntreated sample by GeneChip Operating Software.

Using the statistical criteria described in ‘‘Materialsand Methods,’’ we selected the differentially ex-pressed genes (expression levels up or down for atleast 2.3-fold; Log2 values of 1.2 and 21.2, respec-tively) in response to bestatin andMeJA treatments forfurther analysis. Microarray data analysis revealedthat MeJA treatment caused 1,320 genes showing up-regulation and 1,069 genes showing down-regulation(Fig. 3). Bestatin treatment, on the other hand, caused1,007 genes showingup-regulation and 844 genes show-ing down-regulation (Fig. 3). The microarray data alsorevealed a high percentage of overlap between bestatin-regulated genes andMeJA-regulated genes; 83% of thegenes induced by bestatin were also induced byMeJA,and 66% of the genes repressed by bestatin were alsorepressed by MeJA (Fig. 3).

Further analyses indicated that genes regulated bybestatin and MeJA treatments at all the time pointsshowed significant correlation. Figure 4 shows theresults of 15- and 60-min time points, showing goodcorrelation between the changes of gene transcrip-tional level in response to bestatin and MeJA treat-ments. These analyses not only verified the reliabilityof our microarray experiment but also substantiatedthe notion that the gene expression profile of bestatin-treated plants is similar to that of JA-treated plants.

Functional classification of genes regulated by bothbestatin and JA treatments was carried out using thetools forGeneOntology (GO)categories (http://plexdb.org/index.php andhttps://www.affymetrix.com) andrevised manually, which demonstrated that bestatincanmimic the biological function of JA in the regulationof gene expression (Table I). For example, most of thestress-related genes regulated by MeJA treatment arealso regulated by bestatin treatment. We also noticedthat many genes related to senescence or cell death are

Figure 4. Correlation analysis for genes regulated by bestatin and MeJA treatments at 15-min (A) and 60-min (B) time points,respectively.

Table I. Functional classification of genes regulated by both bestatinand JA treatments using GO categories

Biological ProcessesGene No.

Up-Regulated Down-Regulated

Metabolism 312 197Transcription 71 34Transport 55 20Response to biotic stimulus 50 8Response to abiotic stimulus 20 12Signal transduction 42 14Senescence and cell death 26 1Photosynthesis and light

response8 16

Response to auxin 5 5Other 399 326

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Table II. The list of selected genes regulated by both bestatin and MeJA

Signal Log2 ratio refers to the change in expression level for a transcript between bestatin or MeJA treatments and untreatment. P value representsthe Change P value.

Gene Function AGI

Bestatin Treatment MeJA Treatment

15 min 30 min 60 min 360 min 15 min 30 min 60 min 360 min

Log2

Ratio

P

Value

Log2

Ratio

P

Value

Log2

Ratio

P

Value

Log2

Ratio

P

Value

Log2

Ratio

P

Value

Log2

Ratio

P

Value

Log2

Ratio

P

Value

Log2

Ratio

P

Value

Metabolism

b-Glucosidase At1g75940 3.3 0.004925 22 0.532344 4.5 0.00002 4 0.000068 4.1 0.00002 3.2 0.000023 4.4 0.00002 23.1 0.945978

Lipoxygenase At1g17420 3.7 0.00002 3.7 0.00002 4.4 0.00002 0.6 0.342437 4.8 0.00002 6.2 0.00002 6.7 0.00002 2.5 0.000052

Putative polygalacturonase At2g43870 23.7 0.998349 20.5 0.961585 21.7 0.998168 20.6 0.921063 20.7 0.999226 21.4 0.994067 24 0.996959 22.1 0.995927

Flavonol synthase At5g08640 21.7 0.99997 21.3 0.999948 21.9 0.99998 22.3 0.999977 21.8 0.99998 21.9 0.99998 21.8 0.999954 20.9 0.99996

Putative lipase At2g42690 20.7 0.999308 21 0.99998 21.3 0.99998 22.4 0.99998 20.4 0.999226 20.6 0.999034 22.1 0.99998 21.9 0.99998

Transcription

DREB2A At5g05410 1.9 0.00003 1.9 0.00002 2.5 0.00002 3.4 0.00002 1.9 0.000046 1.6 0.00002 3 0.00002 2.4 0.00002

R2R3-MYB

transcription factor

At3g50060 4.6 0.00002 3.1 0.00002 2.3 0.00004 1.3 0.065566 4.4 0.00002 3.3 0.00002 1 0.00003 1.3 0.004481

WRKY transcription factor At1g80840 6.1 0.00002 4.6 0.00002 4.5 0.00002 5.2 0.00002 5.8 0.00002 5.7 0.00002 6.5 0.00002 5 0.00002

MYB-related

transcription factor

At3g23250 5.8 0.000088 4.7 0.002753 5 0.00002 4.9 0.000241 6.4 0.00002 6.7 0.00003 8.1 0.00002 4.1 0.00712

C2H2-type zinc-finger

protein

At1g68130 20.8 0.994067 21.3 0.998349 20.7 0.995927 21.8 0.99994 20.6 0.997968 21.7 0.999899 21.6 0.99998 20.6 0.99997

Transport

Sugar transporter-like

protein

At4g36670 1 0.330589 2.1 0.000068 3.1 0.00002 1.4 0.000241 1.3 0.000078 3.2 0.00002 3.6 0.00002 1.4 0.000241

Peptide transporter At1g22570 0 0.441923 2.4 0.00002 2.8 0.00002 0 0.493524 1.8 0.00002 2.3 0.00002 1.4 0.00003 0.6 0.366593

Ammonium transporter At3g24290 22.6 0.999759 20.1 0.881991 20.7 0.999922 23.9 0.991489 0 0.921063 21.9 0.99997 22.5 0.998799 23.4 0.999786

Nitrate transporter At3g21670 20.2 0.998923 22 0.99998 22.3 0.99998 23.3 0.99998 20.6 0.999948 20.5 0.999833 23.1 0.99998 21.9 0.99998

Abiotic stress response

Glutathione S-transferase At2g29470 0.8 0.506476 2.8 0.018128 2.7 0.000167 7.6 0.00002 20.3 0.814019 1.7 0.044149 4.4 0.00002 7.3 0.00002

Drought-induced-19-like 1 At4g02200 1.9 0.000023 1.6 0.00002 0.4 0.004481 20.4 0.846768 1.6 0.00002 1.8 0.00002 0.9 0.000492 0.1 0.5

Heat-shock protein At5g52640 0.8 0.002753 0.4 0.161038 2 0.00002 3.8 0.00002 0.2 0.5 0.2 0.5 2.5 0.00002 2.6 0.00002

DRE CRT-binding protein

DREB1C

At4g25470 3.7 0.000023 2.3 0.000101 0.8 0.366593 20.4 0.657563 3.7 0.000023 2.7 0.000046 20.6 0.747149 20.5 0.5

Biotic stress response

Peroxidase At5g19890 1.4 0.013078 1.7 0.00002 1.2 0.000438 0.9 0.003699 0.9 0.001651 2.5 0.00002 2.2 0.00002 1.9 0.000241

Oxidoreductase-like protein At5g38000 1.5 0.000241 20.6 0.868657 2.9 0.00002 2.1 0.001486 2.6 0.000027 2.4 0.00002 2.8 0.00002 1.2 0.001201

Lectin protein At1g65400 5.8 0.00004 5.8 0.00002 6.9 0.00002 4.8 0.000966 4.5 0.00002 6.4 0.00002 7.6 0.00002 4.5 0.00002

Disease resistance protein At2g32140 4.9 0.000241 5 0.001651 3.8 0.000492 5 0.000078 5.7 0.000101 6 0.00004 5.7 0.000046 4 0.000046

Peroxidase At5g64110 20.5 0.99775 22.8 0.995519 22.1 0.999977 21.3 0.998514 21.3 0.999226 21.1 0.999654 21 0.999562 21.9 0.999654

Pathogenesis-related

protein (PR-1)

At2g19990 21 0.996959 20.3 0.532344 20.8 0.999226 24.1 0.99997 21.7 0.99775 20.7 0.975245 23.2 0.999977 20.7 0.988955

Signal transduction

Putative receptor

protein kinase

At1g74360 2 0.001336 1.8 0.011045 2.8 0.000241 5.2 0.00002 1.5 0.094279 2.4 0.010135 4.1 0.00002 3.9 0.00002

Calcineurin B-like protein 1 At4g17615 1 0.105663 1.1 0.000167 1.5 0.00002 2.8 0.00004 1.5 0.000147 2.1 0.00002 2.5 0.000088 1.9 0.00003

Phosphoinositide-specific

phospholipase C

At5g58670 1 0.296089 1 0.035785 2.7 0.00002 1.4 0.001832 0.9 0.000389 2.5 0.00002 4.5 0.00002 1.3 0.000214

Protein phosphatase 2C At1g07160 4.3 0.111714 1.2 0.318909 5.8 0.000307 6.7 0.000966 0.6 0.583567 1 0.185981 6.7 0.000346 6.7 0.000346

Putative receptor

protein kinase

At5g65600 1.7 0.263341 2.9 0.078937 4.3 0.000692 6.6 0.000167 2.8 0.069813 5.5 0.009292 5.8 0.000552 5.8 0.000346

Photosynthesis and

light response

Lhcb3 chlorophyll

a/b-binding protein

At5g54270 2.1 0.00002 2.4 0.00002 0.4 0.000865 0 0.506476 2.5 0.00002 2.7 0.00002 20.4 0.998799 20.7 0.99998

PSII type I chlorophyll

a/b-binding protein

At2g34430 1.4 0.000027 1.4 0.000027 20.2 0.99998 0.2 0.5 1.3 0.00002 1.5 0.00002 21.7 0.99998 20.5 0.999977

Nonphototropic

hypocotyl protein

At3g15570 0 0.938478 21.5 0.999911 24.6 0.99998 25.2 0.99997 20.6 0.997968 21.1 0.99998 25.1 0.99998 22.9 0.99998

Putative chlorophyll

a/b-binding protein

At2g40100 20.7 0.99998 22.1 0.99998 21 0.99998 22 0.99998 21 0.99998 21.6 0.99998 21 0.999977 22 0.99998

Putative nonphototropic

hypocotyl

At1g52770 20.8 0.981872 22.2 0.999759 23.6 0.999922 20.8 0.998923 20.2 0.838962 22.4 0.999693 22.1 0.999727 21 0.997247

Senescence and cell death

Disease resistance protein At1g56510 1.4 0.001201 3.3 0.00002 2.3 0.00002 0.9 0.354442 2.1 0.000023 3.1 0.00002 2.1 0.000101 0.3 0.454766

Disease resistance-like

protein

At5g04720 1.7 0.000027 1.2 0.00002 1.3 0.00002 0.9 0.074268 1.7 0.00002 1.9 0.00002 1.3 0.00003 0.9 0.004073

Senescence-associated

protein sen1

At4g35770 4.4 0.00002 1.2 0.000052 0.8 0.000552 3.5 0.00002 2.2 0.00002 4.9 0.00002 0.6 0.078937 4.1 0.00002

Disease resistance

protein-like

At5g41740 4.5 0.00002 4.9 0.000692 4.8 0.000068 5 0.00002 4.9 0.000088 4.6 0.000068 5.1 0.000114 4 0.006503

Auxin response

Auxin-induced proteins At3g09870 7.6 0.000046 7 0.000241 4.2 0.000774 0.6 0.366593 7.6 0.00002 6.2 0.00002 3.8 0.000774 4.3 0.000438

Putative GH3-like protein At4g03400 0.8 0.026698 2.2 0.00003 2.6 0.00002 20.2 0.938478 0.9 0.000114 3 0.00002 3 0.00003 22 0.999886

auxin-induced protein At1g29510 20.6 0.99751 22.7 0.999308 20.8 0.999448 21.7 0.990708 21.3 0.99987 21.3 0.998349 20.4 0.971234 22.5 0.999786



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induced by bestatin and MeJA treatments, and manygenes related to photosynthesis or light response arerepressed by bestatin andMeJA treatments. The repre-sentative genes regulated by bestatin and MeJA treat-ments are listed in Table II. Expressiondata for all of thedifferentially regulated genes are listed in the sup-plemental data (Supplemental Tables I–III). The highsimilarity of gene transcriptional profiles betweenbestatin- and JA-treated Arabidopsis plants providedfurther support to the hypothesis that bestatin exerts itsbiological function through specific modulation of theJA-signaling pathway.

Isolation of ber Mutants

The above-mentioned unique biological function ofbestatin to specifically modulate the JA-responsepathway prompted us to employ this chemical as anexperimental tool to further dissect JA signaling inArabidopsis using the so-called chemical geneticsapproach, which potentially could circumvent theproblems of gene functional redundancy or lethal mu-tation (Surpin et al., 2005). To this end, we attemptedto screen for ber Arabidopsis mutants as a first step to

elucidate the molecular mechanisms of bestatin actionin plants. In addition to mimicking JA function interms of gene expression, bestatin also stimulatesseveral JA-related developmental phenotypes, includinginhibition of primary root elongation, promotion oflateral root formation, and anthocyanin accumulation(Fig. 5, A–C), which potentially could provide us witha convenient assay to screen for ber mutants. We nextinvestigated the responses of coi1 and other knownJA-related mutants to bestatin treatment in terms ofinhibition of root elongation. Interestingly, the coi1mutant is as responsive as wild type to bestatin interms of inhibition of primary root elongation(Fig. 5D). This result indicates that even though thebestatin-induced defense gene expression strictly de-pends on the COI1 protein function, the bestatin-induced root growth inhibition does not. However,our parallel root growth assay indicated that theJA-insensitive mutant jin1 (Berger et al., 1996; Lorenzoet al., 2004) was significantly less sensitive to bestatinthan the wild type (Fig. 5D). Together, these resultsimply that it is possible to identify genetic loci in-volved in JA signaling through screening for bestatin-response mutants. Based on these observations, we set

Figure 5. The chemical structures of bestatinand JA and their effects on root growthof Arabidopsis seedlings. A, The chemicalstructure of JA and 7-d-old wild-typeArabidopsis (Col-0) seedlings grown on in-dicated concentrations of JA. B, The chem-ical structure of bestatin and 7-d-old Col-0seedlings grown on indicated concentra-tions of bestatin. C, Quantitative analysisof the inhibitory effects on primary rootelongation of bestatin and JA. Root lengthsof seedlings as given in Figure 5, A and B,were measured. Data represent mean 6 SD

of 10 plants per genotype. Similar resultswere obtained from at least three repeatsof this experiment. D, Comparison of rootgrowth phenotypes of Col-0, coi1, and jin1in response to bestatin. Seedlings werephotographed 6 d after germination onMS medium (top) or MS medium contain-ing 25 mM bestatin (bottom).

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up a large-scale genetic screen for Arabidopsis mu-tants that are resistant to the inhibitory effects ofbestatin on primary root elongation. Approximately150,000 Arabidopsis seeds derived from an ethyl-methane sulfonate (EMS)-mutagenized M2 populationwere examined on Murashige and Skoog (MS) me-dium containing 25mMbestatin, and 300 seedlingswithelongated roots were selected as putative bestatin-resistantmutants. Seeds harvested from theprimarypu-tative mutants were rescreened on bestatin-containingmedium, and 19 ber mutants were retained for furtheranalysis.

Response of ber Mutants to JA

The unique biological function of bestatin to mod-ulate JA-signaled processes led us to the hypothesisthat our collection of bestatin-response mutants couldrecover novel genetic loci involved in JA signaling. Totest this possibility, we examined the responses of the

ber mutants to exogenous JA. For these experiments,seeds of each of the 19 ber mutants were further testedseparately on medium containing exogenous JA. Asexpected, we identified ber mutants with altered JAresponse. Based on the root elongation phenotype ofthese ber mutants in response to bestatin and JA, the19 ber mutants were classified into three phenotypicgroups. The first group of ber mutants, which includesber6, ber7, ber188, ber229, ber328, and ber759, is insen-sitive to both bestatin and JA and therefore wasdesignated as JA-insensitive ber mutants (Fig. 6). TheJA-insensitive phenotype of this group of ber mutantssuggests they could define positive regulators in theJA-signaling pathway. The second group containedthree independently isolated mutants: ber157, ber426,and ber544. Because these mutants are insensitive tobestatin but are hypersensitive to JA, they were des-ignated as JA-hypersensitive ber mutants (Fig. 7). Thisgroup of ber mutants could define negative regulatorsin JA signaling. The third and largest group of ber

Figure 6. Root elongation phenotype of the groupof JA-insensitive ber mutants. A, Root phenotypesof Col-0, ber6, ber7, ber188, ber229, ber328, andber759 in response to bestatin and JA. Seedlingswere photographed 7 d after germination on MSmedium and MS medium containing 25 mM

bestatin or 20 mM MeJA. B, The root length wasmeasured for seedlings as given in Figure 6A. Thevalues represent mean 6 SD of 10 plants pergenotype.



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mutants, which includes ber38, ber227, ber281, ber336,ber338, ber369, ber374, ber399, ber581, and ber670, isinsensitive to bestatin but shows normal response toJA (Fig. 8). This group of mutants might define genesinvolved in the uptake, transport, metabolism, and/orperception of bestatin by plants. Taken together, theseresults indicate that we have recovered some JA-related mutants from the ber mutants identified bythe bestatin-based chemical genetic screening.

Genetic and Phenotypic Analyses of ber Mutantswith Altered JA Responses

Further characterization efforts were primarily fo-cused on the two phenotypic groups of ber mutantswith altered JA responses, which may define novel

components in JA signaling. We conducted a set ofcrosses to determine the genetic basis of these bermutants. Each of these nine mutants was backcrossedtowild type (Columbia-0 [Col-0]), and the phenotype ofthe resulting F1 plantswas scored on bestatin-containingmedium and comparedwith those of the parental lines(i.e. mutant and Col-0). F1 plants derived from crossesfor all of the nine mutants (including six JA-insensitiveber mutants and three JA-hypersensitive ber mutants)displayed wild-type phenotype (i.e. normal responseto bestatin in terms of inhibition of root elongation;data not shown). F1 plants were self-pollinated and thephenotype of F2 progeny was scored. The segregationratio of bestatin-sensitive plants:bestatin-resistant plantswas close to 3:1 in all F2 populations (data not shown).These results indicate that the bestatin-resistant phe-notype of each of the nine ber mutants is caused by asingle recessive mutation in a nuclear gene.

Complementation tests among the six lines of JA-insensitive ber mutants revealed that they are notallelic to each other. Test crosses were also conductedbetween each of the six mutants and the reportedJA-insensitivemutants, including jai1/jin1 (Bergeret al.,1996; Lorenzo et al., 2004) and coi1 (Feys et al., 1994;Xie et al., 1998), the results indicated that ber328 isallelic to jin1, which was recently shown to encode abHLH protein called AtMYC2 (Lorenzo et al., 2004),and none of the mutants is allelic to coi1.

Allelism analysis among the three JA-hypersensitiveber mutants demonstrated that each of them representsan independent locus. Each of the three JA-hypersen-sitive ber mutants also displays specific morphologicalphenotypes. For example, ber157 seedlings exhibitstunted growth and short roots without JA treatment(Fig. 7A). Another striking phenotype of ber157 is vires-cence, i.e. young leaves or newly expanded tissues werepale, whereas more mature tissues were as green aswild type (Fig. 9, A and B). Similar to the Arabidopsistransparent testa mutants that are deficient in pigmentaccumulation in the testa (seed coat; Shirley et al., 1995),seeds of the ber426 mutant are yellow or pale brown incolor, easily distinguishable from the brown seeds ofwild-type plants (Fig. 9, C and D). In the absence ofexogenous JA, the ber544 seedlings mimic phenotypesof JA-treated, wild-type plants, i.e. stunted growth andrelatively shorter roots (Fig. 7A). The typical morphol-ogy of ber544 adult plants includes narrow leaf blade,semidwarfism, and small stature (Fig. 9E). To test if thethree JA-hypersensitive mutants define novel loci in JAsignaling or represent new alleles of the reported cev1(Ellis and Turner, 2001) and cex1 (Xu et al., 2001)mutants, which show constitutive expression of JA-related genes, we determined the chromosome locationsof the three genes through standard genetic mapping.BER157 was located on chromosome 4 and linked tomarkers ciw7 and nga1107. BER426 was mapped to aregion between markers nga249 and ciw8 on the shortarm of chromosome 5. BER544 was initially mappedbetweenmarkers CTR1 and nga249 on the short arm ofchromosome 5 and later was delimited to a region

Figure 7. Root elongation phenotype of the group of JA-hypersensitiveber mutants. A, Root phenotypes of Col-0, ber157, ber426, and ber544in response to bestatin and JA. Seedlings were photographed 7 d aftergermination on MS medium and MS medium containing 25 mM bestatinor 20 mM MeJA. B, The root length was measured for seedlings as givenin Figure 7A. The values represent mean 6 SD of 10 plants per genotype.

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containing five BAC clones. To our knowledge, none ofthese loci represents a known genetically identifiedlocus with JA-hypersensitive phenotypes, implyingthat these three JA-hypersensitive ber mutants definenovel genes in JA signaling.

JA-Induced Defense Gene Expression in ber Mutants

To examine the effects of ber mutations on the JA-or wound-response pathway, we further compareddefense-related marker gene expression betweenwild type and representative ber mutants. Seedlingswere either mechanically wounded or treated withJA, and the AtVSP1 transcripts were measured atdifferent time after treatment. Wild-type plants accu-mulated high levels of AtVSP1 transcripts in response

to JA or mechanical wounding (Fig. 10). However, theJA-insensitive ber759 was significantly impaired inJA-induced AtVSP1 transcript accumulation (Fig.10A). In contrast, a JA-hypersensitive ber mutant,ber544, constitutively showed elevated AtVSP1 ex-pression without treatment (Fig. 10, B and C). Whentreated with JA and mechanical wounding, AtVSP1mRNA accumulation levels in this mutant were sig-nificantly higher than those in wild type (Fig. 10, Band C).

DISCUSSION

More and more laboratories are exploring the useof synthetic molecules as tools to solve biological

Figure 8. Root elongation phenotype of the group of ber mutants with normal JA responses. A, Root phenotypes of Col-0, ber38,ber227, ber281, ber336, ber338, ber369, ber374, ber399, ber581, and ber670 in response to bestatin and JA. Seedlings werephotographed 7 d after germination on MS medium and MS medium containing 25 mM bestatin or 20 mM MeJA. B, The root lengthwas measured for seedlings as given in Figure 8A. Data represent mean 6 SD of 10 plants per genotype.



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problems (Schreiber, 2000; Blackwell and Zhao, 2003;Zhaoet al., 2003;Armstronget al., 2004;Vermaetal., 2004;Zouhar et al., 2004; Dai et al., 2005). Among the majorchallenges of using a synthetic compound in a biolog-ical system are to determine the specificity of thecompound in the biological process of interest and todefine the targets and action mechanism of the usedcompound. In general, the specificity of a particularcompound can be determined if (1) the compoundregulates a set of genes known to be involved in theprocess of interest (e.g. this can often be assessed by aDNAmicroarray experiment), (2) the compound causesany known developmental phenotypes related to theprocess of interest, and/or (3) knownmutants involvedin the process respond to the compound as predicted(Blackwell andZhao, 2003).Wehaveprovideda series ofgenetic and molecular evidence showing that bestatinactivates the expression of JA-inducible, defense-related genes in tomato and Arabidopsis (Figs. 1Aand 2, A–C). Furthermore, the action of bestatin toactivate defense genes expression requires the functionof the F-box proteinCOI1/Jai1, a central regulator of JAsignaling (Figs. 1, B and C, and 2, A and B). Microarrayanalysis demonstrates that the gene transcriptionalprofile of bestatin-treated Arabidopsis plants is similarto that of JA-treated plants (Figs. 3 and 4; SupplementalTables I–III). Finally, bestatin promotes several JA-related developmental phenotypes, including inhibi-tion of root growth (Fig. 5). Our data clearly support theconclusion that the action of bestatin to activate defense

genes expression strictly depends on COI1-mediatedJA signaling. However, the fact that the JA-insensitivecoi1 mutant is fully responsive to bestatin treatment interms of root growth inhibition (Fig. 5D) leads to theconclusion that certain JA-related phenotypes trig-gered by bestatin are COI1 independent. One possibleexplanation for this phenomenon is that bestatin mighttarget different proteins to trigger defense gene expres-sion or inhibit root elongation. In contrast to the coi1mutant, our parallel root growth assay indicated thatthe JA-insensitive mutant jin1 (Berger et al., 1996;Lorenzo et al., 2004) was significantly less sensitive tobestatin than the wild type (Fig. 5D). Taken together,these results ledus to thehypothesis that it is possible toidentify genetic loci involved in JA signaling throughscreening for bestatin-response mutants. Using bestatinas a chemical tool, we have applied a chemical geneticapproach to dissect JA signaling, with the hope to

Figure 9. Developmental phenotypes of JA-hypersensitive ber mutants.A and B, Phenotypic comparison between wild type (Col-0) andber157. Shown is 2-week-old wild type (A) and ber157 (B) on MSmedium. The young leaves of ber157 are pale. C and D, Comparison ofseed coat color between wild type and the ber426 mutant. ber426 (C)displays lighter color in seed coat compare to wild type (D). E, Five-week-old wild type (left) and ber544 mutant (right).

Figure 10. JA- and wound-inducedAtVSP1 expression in representativeber mutants showing altered JA response. A, JA-induced AtVSP1 ex-pression is attenuated in the JA-insensitive ber759. Two-week-old Col-0and ber759 mutant seedlings were treated with 50 mM MeJA or nottreated (0). Plant tissues were harvested at indicated times for total RNAextraction. RNA-blot hybridization was performed using 32P-labeledAtVSP1 as probe. A duplicate gel stained with ethidium bromide wasused as RNA loading control. B, JA-induced AtVSP1 expression iselevated in the JA-hypersensitive ber544. Two-week-old Col-0 andber544 seedlings were treated with 50 mM MeJA or not treated (0). Planttissues were harvested 6 h after treatment for total RNA extraction. RNA-blot hybridization was performed using 32P-labeled AtVSP1 as probe. Aduplicate gel stained with ethidium bromide was used as RNA loadingcontrol. C, Wound-induced AtVSP1 expression is elevated in theJA-hypersensitive ber544. Twenty-day-old Col-0 and ber544 seedlingswere wounded with a hemostat or not wounded (0). Plant tissues wereharvested 6 h after treatment for total RNA extraction. RNA-blot hybrid-ization was performed using 32P-labeled AtVSP1 as probe. A duplicategel stained with ethidium bromide was used as RNA loading control.

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circumvent the gene redundancy and mutation le-thality problems, which are intractable by the conven-tional genetic approach. Indeed, our genetic screen forbestatin-resistantArabidopsis plants did lead to identi-fication of two phenotypic groups of mutants withaltered JA responses, i.e. JA-insensitive bermutants andJA-hypersensitive ber mutants. Among the group ofJA-insensitive ber mutants are additional alleles ofknown JA-response mutants (for example, jin1/atmyc2;Lorenzo et al., 2004) andnovel ones.Wenoted thatmostof the JA-insensitive ber mutants show strong insen-sitivity to bestatin but display relatively weak in-sensitivity to JA (Fig. 6), which probably is the reasonwhy these mutants did not come out from previousdirect JA-resistant mutant screens. The strong bestatin-insensitive phenotype of these mutants also providesuswithafacileassaytoidentifythecorrespondinggenesusing a standard map-based cloning method, againshowing the power of the bestatin-based approach toovercome the difficulty caused by gene redundancy inprevious JA studies. Apparently, the identification ofnew components involved in the JA-signaling pathwaydefined by these ber mutants will extend our under-standing of the molecular mechanisms that underlieJA-dependent responses. These results also demon-strated that bestatin-based chemical genetic screeningis a very effective tool to dissect JA signaling inArabidopsis.The other concern of using the chemical genetic

approach to solve biological problems is to identify thesmall molecule targets and to elucidate their actionmechanisms. Recent elegant workwith the small mole-cule Sirtinol in auxin studies has demonstrated thepower of combining genetic studies and chemicalanalyses in determining the action mechanisms of asynthetic compound in the model plant Arabidopsis(Zhao et al., 2003; Dai et al., 2005). Because bestatin andJA are not structurally related (Fig. 5, A and B) butinduce similar responses with respect to defense-related gene expression, we hypothesized that bestatinand JA most likely target different components in JAsignaling and that bestatin targets should be importantcomponents in JA signaling. It is expected that ourgenetic screen for mutants insensitive to the effects ofbestatin should identify genes involved in bestatinuptake, transport, and metabolism, as well as targetsand downstream signaling components. Among these,targets for bestatin are of most interest and signifi-cance. Given our systematic analyses have demon-strated that bestatin, an inhibitor of metalloprotease(Scornik and Botbol, 2001), causes JA responses in to-mato andArabidopsis in a JA biosynthesis-independentbut COI1-dependent manner, this action mode leadsus to the hypothesis that bestatin likely inhibits aprotease that serves as a negative regulator in JA sig-naling. Furthermore, the action mode of bestatin alsosupports the prediction that a mutant plant harboringmutation of bestatin target will be insensitive tobestatin but hypersensitive to JA; this phenotype ex-actly fits the phenotype of our second group of ber

mutants (i.e. JA-hypersensitive ber mutants) that in-cludes ber157, ber544, and ber426. Our ongoing effortsto characterize these mutants, identify the correspond-ing genes, and perform functional analyses will be ofgreat importance for the elucidation of the molecularmechanisms of bestatin biological function and fur-ther understanding of the molecular basis of JAsignaling.

MATERIALS AND METHODS

Plant Materials and Bioassay

Tomato (Lycopersicon esculentum) cv Castlemart was used as the wild-type

cultivar for all experiments involving bioassay of bestatin and JA. The tomato

wound-response mutants spr2 (Li et al., 2003) and jai1-1 (Li et al., 2004) were in

the genetic background of cv Castlemart. Tomato plants were grown at standard

conditions as described previously (Schaller et al., 1995; Li et al., 2003). Eighteen

to 20 d after planting, plants were excised at the base of the stem and placed in

0.5-mL microfuge tubes containing 300 mL of the inducing compound for 2 h.

Plants were then transferred to glass vials containing 20 mL of water and

incubated in a Lucite box for 24 h under continuous light. PI-II levels in leaves

were measured by radial immunodiffusion assay (Ryan, 1967). Briefly, leaves of

the treated seedlings were collected and ground together using a glass pestle on

ceramic to get leave juice. Five microliters of leaf juice was loaded on PIN-II

plates containing tomato PI-II antiserum. After 24 h of incubation on bench top,

the PIN-II plates were developed with 7% acetic acid for 5 min. Diameters (mm)

of the PIN-II radial immunodiffusion rings were measured with a caliper. The

PI-II concentrations (mg/mL leaf juice) were determined using the following

formula: (diameter 3 diameter 2 625) 3 0.016.

JA, MeJA, and bestatin were purchased from Sigma (catalog nos. J2500,

392707, and B8385 respectively), and the inducers were diluted from stock

solutions into sodium phosphate buffer (15 mM sodium phosphate, pH 6.5)

prior to use.

The Col-0 ecotype of Arabidopsis (Arabidopsis thaliana) was used as wild

type. The JA-response mutant coi1 was kindly provided by Dr. Daoxin Xie

(Tsinghua University, Beijing). For JA- and bestatin-response analysis, 2-week-

old seedlings grown on 0.53 MS medium were sprayed with solutions

containing the chemical compounds, and then incubated in growth chamber

under continuous light. Tissues were then harvested at the time intervals as

indicated for RNA extraction. Twenty-day-old Arabidopsis seedlings grown

on 0.53 MS medium were wounded with a hemostat across the midvein of

two expanded leaves. Before wounding or 6 h after wounding, leaves were

harvested for total RNA isolation as described below.

Northern-Blot Analysis

Total RNA was prepared by a guanidine thiocyanate extraction method,

and RNA gel-blot analysis was performed as described previously (Li et al.,

2005). Ten micrograms of total RNAwas separated in an agarose gel contain-

ing 10% formaldehyde, blotted onto a Hybond N1 membrane (Amersham),

and probed with the PCR-amplified DNA fragments using the following primers:

PI-II F (5#-TCAGAAGGAAGTCCGCTAAATC-3#) and PI-II R (5#-TTGCCTTG-GGTTCATCACTCT-3#) for PI-II from tomato, and AtVSP1F (5#-ATGAAAAT-

CCTCTCACTTTCA-3#) and AtVSP1R (5#-TATCCATATTTAGCGTAGTAGG-3#)for AtVSP1 from Arabidopsis (Col-0).

Plasmid Construction and Plant Transformation

A 740-bp fragment upstream of theAtVSP1 coding sequence was isolated by

PCR from genomic DNA (Col-0) using the primers 5#-AAGAAAATCAAGCT-

TTAACCTAAAATCAAC-3# and 5#-GTCGGATCCAGTTTATGGTGTTTATTT-

GTG-3#. The PCR product was digested with HindIII and BamHI and inserted

into the same sites of pBI121 (CLONTECH) to yield the construct AtVSP1::GUS.

The construct was then transformed into Agrobacterium tumefaciens strain

GV3101 (pMP90), whichwas used for transformation of Arabidopsis by vacuum

infiltration (Bechtold et al., 1993). A homozygous transgenic line of AtVSP1::

GUS was identified and used for GUS staining experiment.



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Analysis of GUS Activity

Histochemical staining for GUS activity in transgenic plants was per-

formed as described previously (Jefferson et al., 1987).

Microarray Experiments and Data Analysis

Two-week-old, light-grownArabidopsis seedlingswere sprayedwith 50mM

bestatin or 50mMMeJA and then incubated in growth chamber. Seedlings were

then harvested at different time points (15, 30, 60, and 360 min) after treatment

for total RNA preparation. The untreated seedlings were harvested for total

RNApreparation used as control. Total RNAwas used for preparing probes for

the microarray experiments, which were carried out according to the protocols

provided by the gene chip manufacturer Affymetrix.

The signal intensity data files of scanned probe arrays were analyzed by

GeneChip Operating Software. The one-sidedWilcoxon’s signed rank test was

the statistical method employed to generate the Detection P value, which is

evaluated against defined cutoffs to determine the Detection call. Signal

values were calculated using the one-step Tukey’s biweight estimate, which

assigns a relative measure of abundance to the transcript. Before comparing

two arrays between bestatin- or MeJA-treated samples and untreated sample,

we set the average signal intensity of the array to a target signal of 500 to

scale/normalize the data. The Wilcoxon’s signed rank test was used to com-

pute each Change P value. The signal Log2 ratio was calculated by comparing

each probe pair on the experiment array to the corresponding probe pair on

the baseline array.

The criteria for selecting the significantly differential expressed genes in

response to bestatin or MeJA treatment are as follows. For up-regulated genes,

the criteria are as follows. (1) The signal Detection calls of at least two of the

four time points are Present. (2) The Change call of at least two of the four time

points is Increase. (3) The signal Log2 ratios of at least two of the four time

points are 1.2 (2.3-fold change) or greater. For down-regulated genes, the

criteria are as follows. (1) The signal Detection call of untreated sample is

Present. (2) The Change call of at least two of the four time points is Decrease.

(3) The signal Log2 ratios of at least two of the four time points are 21.2 (2.3-

fold change) or lower. Signal Detection call, Change call, and signal Log2 ratio

are calculated separately using three independent metrics. Change call and

signal Log2 ratio represent the change in expression level for a transcript

between bestatin or MeJA treatments and nontreatment. Correlation analyses

for the changes of gene transcriptional level in response to bestatin and MeJA

treatments were performed in Microsoft Excel. Functional classification of

genes regulated by both bestatin and MeJA treatments was carried out using

the tools for GO categories (http://plexdb.org/index.Php and https://

www.affymetrix.com) and revised manually.

Bestatin-Resistant Mutant Screen and Genetic Analysis

EMS-mutagenized Arabidopsis Col-0 M2 seeds were kindly provided by

Dr. Jianru Zuo (Institute of Genetics and Developmental Biology, Chinese

Academy of Sciences, Beijing). The M2 seeds were germinated and grown on

0.53 MSmedium containing 25mM bestatin underwhite light (16-h-light/8-h-

dark cycle) at 22�C for 8 to 10 d. Seedlingswith elongated rootswere selected as

putative bermutants and directly transplanted to soil. ResultingM3 seeds from

the putative mutants were retested on 25 mM bestatin by measuring root

elongation. The confirmed ber mutants were then tested for their responses to

applied JA. The nine ber mutants showing altered JA responses were back-

crossed at least twice towild type before physiological andmolecular analyses.

For mapping, the ber mutants in Col-0 background were crossed to the

polymorphic ecotype Landsberg erecta, and the resulting F1 plants were self-

pollinated to yield F2 populations segregating for the ber mutant phenotype.

Simple sequence length polymorphism markers were used for linkage anal-

ysis using the standard procedures as described (Lukowitz et al., 2000).

ACKNOWLEDGMENTS

We gratefully acknowledge Dr. Clarence Ryan (Washington State Univer-

sity, Pullman, WA) for providing tomato PI-II antiserum, Dr. Gregg Howe

(Michigan State University, East Lansing, MI) for providing the tomato spr2

and jai1-1 seeds, Dr. Jianru Zuo (Institute of Genetics and Developmental

Biology, Chinese Academy of Sciences, Beijing) for providing EMS-mutagenized

Arabidopsis (Col-0) M2 seeds, Dr. Daoxin Xie (Tsinghua University, Beijing)

for providing coi1 seeds, and Dr. Salome Prat (Institut de Biologia Molecular

de Barcelona) for providing homozygous atmyc2-1 (SALK_040500) and

atmyc2-2 (SALK_083483) seeds. We also thank Wenying Xu and Wenkun

Zhou for their help in microarray data analyses.

Received March 14, 2006; revised June 10, 2006; accepted June 11, 2006;

published June 23, 2006.

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Bestatin, an Inhibitor of Aminopeptidases, Provides a Chemical Genetics Approach to Dissect

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