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First published online June 23, 2006; 10.1104/pp.106.080390

Plant Physiology 141:1400-1413 (2006)
© 2006 American Society of Plant Biologists

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DEVELOPMENT AND HORMONE ACTION

Bestatin, an Inhibitor of Aminopeptidases, Provides a Chemical Genetics Approach to Dissect Jasmonate Signaling 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 Genetics and 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 Academy of 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


   ABSTRACT
TOP
 ABSTRACT
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 LITERATURE CITED
 
Bestatin, a potent inhibitor of some aminopeptidases, was shown previously to be a powerful inducer of wound-response genes in tomato (Lycopersicon esculentum). Here, we present several lines of evidence showing that bestatin specifically activates jasmonic acid (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-signaling pathway, but does not depend strictly on JA biosynthesis. Third, microarray analysis using Arabidopsis whole-genome chip demonstrates that the gene expression profile of bestatin-treated plants is similar to that of JA-treated plants. Fourth, bestatin promotes 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 JA signaling. We have employed bestatin as an experimental tool to dissect JA signaling through a chemical genetic screening, which yielded a collection of Arabidopsis bestatin-resistant (ber) mutants that are insensitive to the inhibitory effects of bestatin on root elongation. 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 ber mutants, and mutants insensitive to bestatin but showing normal response to JA. Genetic and phenotypic analyses of the ber mutants 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 activating the expression of genes involved in herbivore deterrence, wound healing, and other defense-related processes. A fascinating feature of induced defense responses is their occurrence both locally at the site of wounding and systemically in undamaged parts throughout the plant (Green and Ryan, 1972Go). Wound-inducible proteinase inhibitors (PIs) in tomato (Lycopersicon esculentum) provide an attractive model system in which to identify the proposed wound signals for defense genes expression. Among the identified candidate signals that can activate wound-induced PIs expression in tomato are systemin, an 18-amino acid peptide, and jasmonic acid (JA), an oxylipin-derived phytohormone (Farmer and Ryan, 1990Go, 1992Go; Pearce et al., 1991Go; Constabel et al., 1995Go; Bowles, 1998Go; Walling, 2000Go; Leon et al., 2001Go; Browse, 2005Go). A wealth of evidence indicates that systemin and JA actually work together in the same signaling pathway to promote systemic expression of PIs and other defense-related genes (Constabel et al., 1995Go; Bowles, 1998Go). However, relatively little is known about the specific roles of systemin and JA in long-distance wound signaling. Recently, a series of grafting experiments conducted with tomato mutants defective in JA biosynthesis or signaling and, as a consequence, compromised in systemic defense responses led to the current notion that systemin acts at or near the site of wounding (locally) to amplify the production of JA, which in turn acts as a long-distance wound signal to promote defense genes expression (Li et al., 2002Go; Ryan and Moura, 2002Go; Turner et al., 2002Go; Stratmann, 2003Go; Howe, 2004Go; Schilmiller and Howe, 2005Go). These studies challenge the previous paradigm that systemin is the sole long-distance wound signal and ascribe JA a central role in regulating systemic defense responses in virtually all species throughout the plant kingdom. In addition to defense, the jasmonate family of signaling molecules, including JA, methyl JA (MeJA), and other bioactive derivatives (collectively referred to here as jasmonates), also play important roles in regulating developmental processes such as root growth, tuberization, senescence, and reproduction (Creelman and Mullet, 1997Go; Turner et al., 2002Go; Wasternack and Hause, 2002Go; Devoto and Turner, 2003Go; Browse, 2005Go). In spite of the importance of jasmonates as stress and growth regulators is well recognized, relatively little is known about the molecular mechanisms governing JA perception and subsequent signaling events leading to activation of downstream JA-responsive genes.

Conventional forward genetic screens for JA-resistant (insensitive) mutants have led to the identification of a few molecular players in the JA-signaling network, including ubiquitin-mediated proteolysis machinery (Turner et al., 2002Go; Devoto and Turner, 2003Go; Browse, 2005Go). A significant advance in our understanding of JA signaling came from the analysis of the Arabidopsis (Arabidopsis thaliana) coronatine insensitive 1 (coi1) mutant that is insensitive to JA. The identification of COI1 encoding an F-box protein (Xie et al., 1998Go) and the demonstration that COI1 interacts with Skp1 and Cullin1 to assemble a functional SCFCOI1 ubiquitin ligase complex in vivo (Devoto et al., 2002Go; Xu et al., 2002Go) suggest that a ubiquitin-mediated protein degradation machinery is involved in JA signaling. Significantly, it was demonstrated that loss of function of a tomato homolog of COI1 (LeCOI1) results in a JA-insensitive phenotype both in terms of defense response and development, suggesting the existence of a COI1 function is conserved among plant species other than Arabidopsis (Li et al., 2004Go). Moreover, plants deficient in the general regulators of SCF complexes, such as Cullin1 (Ren et al., 2005Go), SGT1b (Gray et al., 2003Go), AXR1 (Leyser et al., 1993Go), and CSN (Feng et al., 2003Go), also show impaired JA responses. Taken together, accumulating evidence shows that the ubiquitin-mediated protein degradation process, which also has been demonstrated in many different hormone-signaling pathways, is a central regulatory mechanism in JA signaling.

By classic forward genetic screening, other important components of the JA-response pathway have also been identified. Compared with coi1, the JA-resistant mutants jar1 (Staswick et al., 1992Go) and jin1 (Berger et al., 1996Go) show a relatively weak phenotype in response to JA. JAR1 encodes an enzyme that has JA adenylation activity to form JA-amino acid conjugates, suggesting covalent modification of JA plays a key role in its action (Staswick et al., 2002Go). JIN1 encodes a nuclear-localized bHLH-type transcription factor known as AtMYC2 (Lorenzo et al., 2004Go). The tomato homologs of AtMYC2, named JAMYC2 and JAMYC10, specifically recognize a T/G box in the promoters of JA-responsive genes and activate their expression (Boter et al., 2004Go). To identify negative regulators of JA signaling, several groups conducted genetic screens for constitutive JA-response mutants (Ellis and Turner, 2001Go; Hilpert et al., 2001Go; Xu et al., 2001Go). To date, only one of these genes (CEV1) has been identified. The finding that CEV1 encodes a cellulose synthase suggests a link between cell wall biosynthesis and JA signaling (Ellis et al., 2002Go).

However, compared to the five classic and other plant hormones, our knowledge of the JA-signaling pathway is still very limited. For example, extensive conventional forward genetic screens failed to identify the JA receptor(s) and the substrate(s) of the SCFCOI1 ubiquitin ligase complex, two of the most critical and apparent gaps in the understanding of JA signaling. The inability to identify the JA receptor(s), SCFCOI1 substrates, and other components in forward genetic screens suggests functional redundancy or lethal mutation in the JA perception and other signaling apparatus. With this in mind, we are looking for an alternative approach that may complement the limitations of conventional genetics to further dissect JA signaling. So-called "chemical genetics" is a powerful approach to dissect biological processes that may be intractable using conventional genetics because of gene function redundancy or lethality (Surpin et al., 2005Go). Chemical genetics is centered around the tenet that small organic molecules can be used like mutations in classic genetics to modulate protein functions and to assist in delineation of biological pathways (Blackwell and Zhao, 2003Go). Once small molecules that affect (inhibit or activate) a biological process of interest are identified, integration of the small molecule-based forward chemical genetics with the ever-growing genomics tools in model plant system (for example, Arabidopsis) will significantly facilitate the identification of relevant gene products, including targets of the active compounds, which are intractable by the classic genetics approach. Indeed, recent work has demonstrated the feasibility of this approach in studies of several biological pathways, including auxin signaling (Blackwell and Zhao, 2003Go; Zhao et al., 2003Go; Armstrong et al., 2004Go; Dai et al., 2005Go), brassinosteroid signaling (Asami et al., 2003Go), vacuole sorting (Zouhar et al., 2004Go), endomembrane trafficking, and gravitropism (Surpin et al., 2005Go).

To identify new components in JA signaling, we have developed a forward chemical genetic screen, which combines the advantages of chemical genetics and the classic genetic screen, to identify novel mutants with altered JA responses. Bestatin, a potent inhibitor of some aminopeptidases in plants and animals, was shown previously to be a powerful inducer of wound-response genes in tomato (Schaller et al., 1995Go). Bestatin has been widely used as an experimental tool in elucidating the physiological role of some mammalian exopeptidases in the regulation of the immune system, in the growth of tumors, and in the degradation of cellular proteins (Scornik and Botbol, 2001Go). We have conducted a series of genetic and molecular analyses in tomato and Arabidopsis, two of the most extensively studied model systems for JA responses, showing that bestatin specifically modulates JA signaling. Bestatin specifically activates the expression of JA-inducible defense-related genes, and the induction kinetics of these genes in response to bestatin are similar to those in response to JA. Furthermore, the action of bestatin to activate defense gene expression requires the function of the F-box protein COI1/Jai1, a central regulator of JA-signaling, but does not depend strictly on JA biosynthesis. Microarray analysis using Arabidopsis whole-genome chip demonstrates that the gene transcriptional profile of bestatin-treated plants is similar to that of JA-treated plants. Finally, bestatin promotes several JA-related developmental phenotypes. Taken together, the unique action mode of bestatin in regulating JA-signaled processes leads us to the hypothesis that bestatin probably exerts its effects by modulating the JA-signaling pathway. To test this hypothesis and, moreover, to identify novel components involved in JA responses, we have employed bestatin as an experimental tool to dissect JA signaling using a chemical genetics approach, which yielded a collection of Arabidopsis bestatin-resistant (ber) mutants insensitive to the inhibitory effects of bestatin on primary root elongation. Further characterization efforts demonstrate that some ber mutants are defective in various JA- and wound-induced responses, which allowed us to classify them into three phenotypic groups: mutants insensitive to both bestatin and JA, mutants insensitive to bestatin but hypersensitive to JA, and mutants insensitive to bestatin but showing normal response to JA. Among the three groups of mutants, the second group, which is insensitive to bestatin but is hypersensitive to JA, is distinct from other identified JA-related mutants. Our ongoing effort to identify the gene functions defined by these novel ber mutants promises to shed new light on the molecular basis of JA signaling and on the molecular mechanisms of the biological function of bestatin in plants.


   RESULTS
TOP
 ABSTRACT
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 LITERATURE CITED
 

Bestatin Specifically Activates JA Signaling in Tomato and Arabidopsis

It was previously reported that bestatin is a powerful inducer of tomato defense genes, which also are induced by herbivore attack, mechanical wounding, and the potent wound-response elicitors, such as systemin and JA (Schaller et al., 1995Go). Given that JA works as a systemic wound signal and plays a central role in regulating defense genes expression in response to herbivore attack and mechanical wounding, we further compared the action mode of bestatin with that of JA in tomato and Arabidopsis, two of the most extensively studied systems on JA and wound signaling. The induction kinetics of proteinase inhibitor II (PI-II) mRNA, a well-known representative marker of a group of wound-inducible mRNAs in tomato, in response to exogenous bestatin and JA, was found to be similar (Fig. 1A ). Significant increase in PI-II mRNA levels was initially observed 2 h after feeding plants with both bestatin and JA, and mRNA abundance continued to rise during the time course, up to 24 h (Fig. 1A).


Figure 1
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  		Figure 1. Activation of JA-inducible genes by bestatin in tomato. A, Induction of PI-II mRNA by bestatin and JA. RNA was isolated from wild-type (WT) tomato leaves at the times shown after treatment of the cutting stems with bestatin (40 nmol/plant, dissolved in 15 mM phosphate buffer), JA (20 nmol/plant, dissolved in 15 mM phosphate buffer), or buffer control (15 mM phosphate buffer). RNA-blot hybridization was performed using 32P-labeled cDNAs for PI-II. A duplicate gel stained with ethidium bromide was used as RNA loading control. B, Effects of the spr2 and jai1-1 mutations on bestatin- and JA-induced PI-II mRNA accumulation. Eighteen-day-old wild-type, spr2, and jai1-1 plants were treated with bestatin, JA, and buffer control as described in A. Twelve hours after treatment, RNA was isolated from leaves of each genotype and probed with 32P-labeled PI-II cDNA. A duplicate gel stained with ethidium bromide was used as RNA loading control. C, Effects of the spr2 and jai1-1 mutations on bestatin- and JA-induced PI-II protein accumulation. Eighteen-day-old wild-type, spr2, and jai1-1 plants were treated with buffer control (white bars), JA (black bars), and bestatin (gray bars), as described in A. Twenty-four hours after treatment, PI-II protein levels were measured. Values represent mean ± SD of 12 plants per genotype.

 
To examine whether the action of bestatin to activate the expression of JA-related defense genes is ubiquitous, we investigated the action of bestatin in Arabidopsis. Treatment of wild-type plants with exogenous bestatin readily induced enhanced expression of VEGETATIVE STORAGE PROTEIN1 (AtVSP1; At5g24780), a widely used marker gene of JA responses in Arabidopsis (Fig. 2B ). Moreover, the induction kinetics of AtVSP1 transcript in response to bestatin is very similar to that in response to JA (Fig. 2, A and B). We further constructed a JA reporter line in which the promoter of the JA-inducible AtVSP1 gene was fused with the beta-glucuronidase (GUS) reporter. Bestatin induces the expression of AtVSP1-GUS throughout the plant, with highest expression in young leaves (Fig. 2E). Moreover, the GUS expression pattern of the JA 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 signaling both in tomato and Arabidopsis.


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

 

The Action of Bestatin to Activate JA Signaling Strictly Depends on COI1 Function

To determine whether bestatin-induced defense gene expression requires a functional JA-signaling pathway, we further compared the capacity of exogenous bestatin and JA to activate JA-inducible marker genes in tomato and Arabidopsis mutants that are known 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., 2003Go), induced the accumulation of PI-II protein to levels comparable to those in wild-type plant (Fig. 1C). However, bestatin failed to induce PI-II accumulation in the jai1-1 plant (Fig. 1C), which is a loss-of-function mutant of the tomato homolog of COI1, an F-box protein required for JA signaling (Li et al., 2004Go). The capacity of bestatin to induce PI-II protein accumulation in wild-type, spr2, and jai1-1 plants is very similar to that of JA, as shown in our parallel experiments (Fig. 1C). We also examined the response of spr2 and jai1-1 to exogenous bestatin at the level of PI-II transcript accumulation. For these experiments, PI-II transcript levels were measured 12 h after treatment with bestatin or JA, and the results obtained were in good agreement with the measurements of PI-II protein levels. Both bestatin and JA induced the accumulation of PI-II transcripts in wild-type and spr2 plants, but not in jai1-1 plants (Fig. 1B). These results demonstrate that the bestatin-induced PI-II accumulation in tomato leaves requires the Jai1/COI1-dependent JA-signaling pathway, but does not depend on JA biosynthesis. Consistent with the results obtained from the tomato system, both bestatin and JA induce AtVSP1 expression in wild-type Arabidopsis plants but not in coi1 plants (Fig. 2, A and B), a well-known JA-signaling mutant in Arabidopsis (Feys et al., 1994Go; Xie et al., 1998Go). These results indicate that the induction of AtVSP1 expression by bestatin, just like by JA, also occurs through the COI1-dependent JA-signaling pathway in Arabidopsis. Taken together, our results indicate that bestatin promotes plant defense responses by activating the JA-signaling pathway, and further suggest that bestatin might exert its action downstream of JA biosynthesis but upstream of the F-box protein COI1.


Gene Transcriptional Profile of Bestatin-Treated Arabidopsis Plants Is Similar to That of JA-Treated Plants

To gain a deeper understanding on the action mechanism of bestatin, we performed a microarray analysis using the Arabidopsis whole-genome chip (Affymetrix) to compare the global gene transcriptional profiles regulated by bestatin and MeJA in wild-type Arabidopsis seedlings. The arrays of bestatin- or MeJA-treated samples at the four time points tested (15, 30, 60, and 360 min) were compared with that of untreated sample by GeneChip Operating Software.

Using the statistical criteria described in "Materials and Methods," we selected the differentially expressed genes (expression levels up or down for at least 2.3-fold; Log2 values of 1.2 and –1.2, respectively) in response to bestatin and MeJA treatments for further analysis. Microarray data analysis revealed that MeJA treatment caused 1,320 genes showing up-regulation and 1,069 genes showing down-regulation (Fig. 3 ). Bestatin treatment, on the other hand, caused 1,007 genes showing up-regulation and 844 genes showing down-regulation (Fig. 3). The microarray data also revealed a high percentage of overlap between bestatin-regulated genes and MeJA-regulated genes; 83% of the genes induced by bestatin were also induced by MeJA, and 66% of the genes repressed by bestatin were also repressed by MeJA (Fig. 3).


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

 
Further analyses indicated that genes regulated by bestatin and MeJA treatments at all the time points showed significant correlation. Figure 4 shows the results of 15- and 60-min time points, showing good correlation between the changes of gene transcriptional level in response to bestatin and MeJA treatments. These analyses not only verified the reliability of our microarray experiment but also substantiated the notion that the gene expression profile of bestatin-treated plants is similar to that of JA-treated plants.


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

 
Functional classification of genes regulated by both bestatin and JA treatments was carried out using the tools for Gene Ontology (GO) categories (http://plexdb.org/index.php and https://www.affymetrix.com) and revised manually, which demonstrated that bestatin can mimic the biological function of JA in the regulation of gene expression (Table I ). For example, most of the stress-related genes regulated by MeJA treatment are also regulated by bestatin treatment. We also noticed that many genes related to senescence or cell death are induced by bestatin and MeJA treatments, and many genes related to photosynthesis or light response are repressed by bestatin and MeJA treatments. The representative genes regulated by bestatin and MeJA treatments are listed in Table II . Expression data for all of the differentially regulated genes are listed in the supplemental data (Supplemental Tables I–III). The high similarity of gene transcriptional profiles between bestatin- and JA-treated Arabidopsis plants provided further support to the hypothesis that bestatin exerts its biological function through specific modulation of the JA-signaling pathway.


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

 

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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 represents the Change P value.

 

Isolation of ber Mutants

The above-mentioned unique biological function of bestatin to specifically modulate the JA-response pathway prompted us to employ this chemical as an experimental tool to further dissect JA signaling in Arabidopsis using the so-called chemical genetics approach, which potentially could circumvent the problems of gene functional redundancy or lethal mutation (Surpin et al., 2005Go). To this end, we attempted to screen for ber Arabidopsis mutants as a first step to elucidate the molecular mechanisms of bestatin action in plants. In addition to mimicking JA function in terms of gene expression, bestatin also stimulates several JA-related developmental phenotypes, including inhibition of primary root elongation, promotion of lateral root formation, and anthocyanin accumulation (Fig. 5, A–C ), which potentially could provide us with a convenient assay to screen for ber mutants. We next investigated the responses of coi1 and other known JA-related mutants to bestatin treatment in terms of inhibition of root elongation. Interestingly, the coi1 mutant is as responsive as wild type to bestatin in terms of inhibition of primary root elongation (Fig. 5D). This result indicates that even though the bestatin-induced defense gene expression strictly depends on the COI1 protein function, the bestatin-induced root growth inhibition does not. However, our parallel root growth assay indicated that the JA-insensitive mutant jin1 (Berger et al., 1996Go; Lorenzo et al., 2004Go) was significantly less sensitive to bestatin than the wild type (Fig. 5D). Together, these results imply that it is possible to identify genetic loci involved in JA signaling through screening for bestatin-response mutants. Based on these observations, we set up a large-scale genetic screen for Arabidopsis mutants that are resistant to the inhibitory effects of bestatin on primary root elongation. Approximately 150,000 Arabidopsis seeds derived from an ethylmethane sulfonate (EMS)-mutagenized M2 population were examined on Murashige and Skoog (MS) medium containing 25 µM bestatin, and 300 seedlings with elongated roots were selected as putative bestatin-resistant mutants. Seeds harvested from the primary putative mutants were rescreened on bestatin-containing medium, and 19 ber mutants were retained for further analysis.


Figure 5
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  		Figure 5. The chemical structures of bestatin and JA and their effects on root growth of Arabidopsis seedlings. A, The chemical structure of JA and 7-d-old wild-type Arabidopsis (Col-0) seedlings grown on indicated concentrations of JA. B, The chemical structure of bestatin and 7-d-old Col-0 seedlings grown on indicated concentrations of bestatin. C, Quantitative analysis of the inhibitory effects on primary root elongation of bestatin and JA. Root lengths of seedlings as given in Figure 5, A and B, were measured. Data represent mean ± SD of 10 plants per genotype. Similar results were obtained from at least three repeats of this experiment. D, Comparison of root growth phenotypes of Col-0, coi1, and jin1 in response to bestatin. Seedlings were photographed 6 d after germination on MS medium (top) or MS medium containing 25 µM bestatin (bottom).

 

Response of ber Mutants to JA

The unique biological function of bestatin to modulate JA-signaled processes led us to the hypothesis that our collection of bestatin-response mutants could recover novel genetic loci involved in JA signaling. To test 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 tested separately on medium containing exogenous JA. As expected, we identified ber mutants with altered JA response. Based on the root elongation phenotype of these ber mutants in response to bestatin and JA, the 19 ber mutants were classified into three phenotypic groups. The first group of ber mutants, which includes ber6, ber7, ber188, ber229, ber328, and ber759, is insensitive to both bestatin and JA and therefore was designated as JA-insensitive ber mutants (Fig. 6 ). The JA-insensitive phenotype of this group of ber mutants suggests they could define positive regulators in the JA-signaling pathway. The second group contained three independently isolated mutants: ber157, ber426, and ber544. Because these mutants are insensitive to bestatin but are hypersensitive to JA, they were designated as JA-hypersensitive ber mutants (Fig. 7 ). This group of ber mutants could define negative regulators in JA signaling. The third and largest group of ber mutants, which includes ber38, ber227, ber281, ber336, ber338, ber369, ber374, ber399, ber581, and ber670, is insensitive to bestatin but shows normal response to JA (Fig. 8 ). This group of mutants might define genes involved in the uptake, transport, metabolism, and/or perception of bestatin by plants. Taken together, these results indicate that we have recovered some JA-related mutants from the ber mutants identified by the bestatin-based chemical genetic screening.


Figure 6
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  		Figure 6. Root elongation phenotype of the group of JA-insensitive ber mutants. A, Root phenotypes of Col-0, ber6, ber7, ber188, ber229, ber328, and ber759 in response to bestatin and JA. Seedlings were photographed 7 d after germination on MS medium and MS medium containing 25 µM bestatin or 20 µM MeJA. B, The root length was measured for seedlings as given in Figure 6A. The values represent mean ± SD of 10 plants per genotype.

 

Figure 7
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  		Figure 7. Root elongation phenotype of the group of JA-hypersensitive ber mutants. A, Root phenotypes of Col-0, ber157, ber426, and ber544 in response to bestatin and JA. Seedlings were photographed 7 d after germination on MS medium and MS medium containing 25 µM bestatin or 20 µM MeJA. B, The root length was measured for seedlings as given in Figure 7A. The values represent mean ± SD of 10 plants per genotype.

 

Figure 8
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  		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 were photographed 7 d after germination on MS medium and MS medium containing 25 µM bestatin or 20 µM MeJA. B, The root length was measured for seedlings as given in Figure 8A. Data represent mean ± SD of 10 plants per genotype.

 

Genetic and Phenotypic Analyses of ber Mutants with Altered JA Responses

Further characterization efforts were primarily focused on the two phenotypic groups of ber mutants with altered JA responses, which may define novel components in JA signaling. We conducted a set of crosses to determine the genetic basis of these ber mutants. Each of these nine mutants was backcrossed to wild type (Columbia-0 [Col-0]), and the phenotype of the resulting F1 plants was scored on bestatin-containing medium and compared with those of the parental lines (i.e. mutant and Col-0). F1 plants derived from crosses for all of the nine mutants (including six JA-insensitive ber mutants and three JA-hypersensitive ber mutants) displayed wild-type phenotype (i.e. normal response to bestatin in terms of inhibition of root elongation; data not shown). F1 plants were self-pollinated and the phenotype of F2 progeny was scored. The segregation ratio of bestatin-sensitive plants:bestatin-resistant plants was close to 3:1 in all F2 populations (data not shown). These results indicate that the bestatin-resistant phenotype of each of the nine ber mutants is caused by a single recessive mutation in a nuclear gene.

Complementation tests among the six lines of JA-insensitive ber mutants revealed that they are not allelic to each other. Test crosses were also conducted between each of the six mutants and the reported JA-insensitive mutants, including jai1/jin1 (Berger et al., 1996Go; Lorenzo et al., 2004Go) and coi1 (Feys et al., 1994Go; Xie et al., 1998Go), the results indicated that ber328 is allelic to jin1, which was recently shown to encode a bHLH protein called AtMYC2 (Lorenzo et al., 2004Go), and none of the mutants is allelic to coi1.

Allelism analysis among the three JA-hypersensitive ber mutants demonstrated that each of them represents an independent locus. Each of the three JA-hypersensitive ber mutants also displays specific morphological phenotypes. For example, ber157 seedlings exhibit stunted growth and short roots without JA treatment (Fig. 7A). Another striking phenotype of ber157 is virescence, i.e. young leaves or newly expanded tissues were pale, whereas more mature tissues were as green as wild type (Fig. 9, A and B ). Similar to the Arabidopsis transparent testa mutants that are deficient in pigment accumulation in the testa (seed coat; Shirley et al., 1995Go), seeds of the ber426 mutant are yellow or pale brown in color, easily distinguishable from the brown seeds of wild-type plants (Fig. 9, C and D). In the absence of exogenous JA, the ber544 seedlings mimic phenotypes of JA-treated, wild-type plants, i.e. stunted growth and relatively shorter roots (Fig. 7A). The typical morphology of ber544 adult plants includes narrow leaf blade, semidwarfism, and small stature (Fig. 9E). To test if the three JA-hypersensitive mutants define novel loci in JA signaling or represent new alleles of the reported cev1 (Ellis and Turner, 2001Go) and cex1 (Xu et al., 2001Go) mutants, which show constitutive expression of JA-related genes, we determined the chromosome locations of the three genes through standard genetic mapping. BER157 was located on chromosome 4 and linked to markers ciw7 and nga1107. BER426 was mapped to a region between markers nga249 and ciw8 on the short arm of chromosome 5. BER544 was initially mapped between markers CTR1 and nga249 on the short arm of chromosome 5 and later was delimited to a region containing five BAC clones. To our knowledge, none of these loci represents a known genetically identified locus with JA-hypersensitive phenotypes, implying that these three JA-hypersensitive ber mutants define novel genes in JA signaling.


Figure 9
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  		Figure 9. Developmental phenotypes of JA-hypersensitive ber mutants. A and B, Phenotypic comparison between wild type (Col-0) and ber157. Shown is 2-week-old wild type (A) and ber157 (B) on MS medium. The young leaves of ber157 are pale. C and D, Comparison of seed 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).

 

JA-Induced Defense Gene Expression in ber Mutants

To examine the effects of ber mutations on the JA- or wound-response pathway, we further compared defense-related marker gene expression between wild type and representative ber mutants. Seedlings were either mechanically wounded or treated with JA, and the AtVSP1 transcripts were measured at different time after treatment. Wild-type plants accumulated high levels of AtVSP1 transcripts in response to JA or mechanical wounding (Fig. 10 ). However, the JA-insensitive ber759 was significantly impaired in JA-induced AtVSP1 transcript accumulation (Fig. 10A). In contrast, a JA-hypersensitive ber mutant, ber544, constitutively showed elevated AtVSP1 expression without treatment (Fig. 10, B and C). When treated with JA and mechanical wounding, AtVSP1 mRNA accumulation levels in this mutant were significantly higher than those in wild type (Fig. 10, B and C).


Figure 10
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  		Figure 10. JA- and wound-induced AtVSP1 expression in representative ber mutants showing altered JA response. A, JA-induced AtVSP1 expression is attenuated in the JA-insensitive ber759. Two-week-old Col-0 and ber759 mutant seedlings were treated with 50 µM MeJA or not treated (0). Plant tissues were harvested at indicated times for total RNA extraction. RNA-blot hybridization was performed using 32P-labeled AtVSP1 as probe. A duplicate gel stained with ethidium bromide was used as RNA loading control. B, JA-induced AtVSP1 expression is elevated in the JA-hypersensitive ber544. Two-week-old Col-0 and ber544 seedlings were treated with 50 µM MeJA or not treated (0). Plant tissues were harvested 6 h after treatment for total RNA extraction. RNA-blot hybridization was performed using 32P-labeled AtVSP1 as probe. A duplicate gel stained with ethidium bromide was used as RNA loading control. C, Wound-induced AtVSP1 expression is elevated in the JA-hypersensitive ber544. Twenty-day-old Col-0 and ber544 seedlings were wounded with a hemostat or not wounded (0). Plant tissues were harvested 6 h after treatment for total RNA extraction. RNA-blot hybridization was performed using 32P-labeled AtVSP1 as probe. A duplicate gel stained with ethidium bromide was used as RNA loading control.

 

   DISCUSSION
TOP
 ABSTRACT
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 LITERATURE CITED
 
More and more laboratories are exploring the use of synthetic molecules as tools to solve biological problems (Schreiber, 2000Go; Blackwell and Zhao, 2003Go; Zhao et al., 2003Go; Armstrong et al., 2004Go; Verma et al., 2004Go; Zouhar et al., 2004Go; Dai et al., 2005Go). Among the major challenges of using a synthetic compound in a biological system are to determine the specificity of the compound in the biological process of interest and to define the targets and action mechanism of the used compound. In general, the specificity of a particular compound can be determined if (1) the compound regulates a set of genes known to be involved in the process of interest (e.g. this can often be assessed by a DNA microarray experiment), (2) the compound causes any known developmental phenotypes related to the process of interest, and/or (3) known mutants involved in the process respond to the compound as predicted (Blackwell and Zhao, 2003Go). We have provided a series of genetic and molecular evidence showing that bestatin activates the expression of JA-inducible, defense-related genes in tomato and Arabidopsis (Figs. 1A and 2, A–C). Furthermore, the action of bestatin to activate defense genes expression requires the function of the F-box protein COI1/Jai1, a central regulator of JA signaling (Figs. 1, B and C, and 2, A and B). Microarray analysis demonstrates that the gene transcriptional profile of bestatin-treated Arabidopsis plants is similar to that of JA-treated plants (Figs. 3 and 4; Supplemental Tables I–III). Finally, bestatin promotes several JA-related developmental phenotypes, including inhibition of root growth (Fig. 5). Our data clearly support the conclusion that the action of bestatin to activate defense genes expression strictly depends on COI1-mediated JA signaling. However, the fact that the JA-insensitive coi1 mutant is fully responsive to bestatin treatment in terms of root growth inhibition (Fig. 5D) leads to the conclusion that certain JA-related phenotypes triggered by bestatin are COI1 independent. One possible explanation for this phenomenon is that bestatin might target different proteins to trigger defense gene expression or inhibit root elongation. In contrast to the coi1 mutant, our parallel root growth assay indicated that the JA-insensitive mutant jin1 (Berger et al., 1996Go; Lorenzo et al., 2004Go) was significantly less sensitive to bestatin than the wild type (Fig. 5D). Taken together, these results led us to the hypothesis that it is possible to identify genetic loci involved in JA signaling through screening for bestatin-response mutants. Using bestatin as a chemical tool, we have applied a chemical genetic approach to dissect JA signaling, with the hope to circumvent the gene redundancy and mutation lethality problems, which are intractable by the conventional genetic approach. Indeed, our genetic screen for bestatin-resistant Arabidopsis plants did lead to identification of two phenotypic groups of mutants with altered JA responses, i.e. JA-insensitive ber mutants and JA-hypersensitive ber mutants. Among the group of JA-insensitive ber mutants are additional alleles of known JA-response mutants (for example, jin1/atmyc2; Lorenzo et al., 2004Go) and novel ones. We noted that most of the JA-insensitive ber mutants show strong insensitivity to bestatin but display relatively weak insensitivity to JA (Fig. 6), which probably is the reason why these mutants did not come out from previous direct JA-resistant mutant screens. The strong bestatin-insensitive phenotype of these mutants also provides us with a facile assay to identify the corresponding genes using a standard map-based cloning method, again showing the power of the bestatin-based approach to overcome the difficulty caused by gene redundancy in previous JA studies. Apparently, the identification of new components involved in the JA-signaling pathway defined by these ber mutants will extend our understanding of the molecular mechanisms that underlie JA-dependent responses. These results also demonstrated that bestatin-based chemical genetic screening is a very effective tool to dissect JA signaling in Arabidopsis.

The other concern of using the chemical genetic approach to solve biological problems is to identify the small molecule targets and to elucidate their action mechanisms. Recent elegant work with the small molecule Sirtinol in auxin studies has demonstrated the power of combining genetic studies and chemical analyses in determining the action mechanisms of a synthetic compound in the model plant Arabidopsis (Zhao et al., 2003Go; Dai et al., 2005Go). Because bestatin and JA are not structurally related (Fig. 5, A and B) but induce similar responses with respect to defense-related gene expression, we hypothesized that bestatin and JA most likely target different components in JA signaling and that bestatin targets should be important components in JA signaling. It is expected that our genetic screen for mutants insensitive to the effects of bestatin should identify genes involved in bestatin uptake, transport, and metabolism, as well as targets and downstream signaling components. Among these, targets for bestatin are of most interest and significance. Given our systematic analyses have demonstrated that bestatin, an inhibitor of metalloprotease (Scornik and Botbol, 2001Go), causes JA responses in tomato and Arabidopsis in a JA biosynthesis-independent but COI1-dependent manner, this action mode leads us to the hypothesis that bestatin likely inhibits a protease that serves as a negative regulator in JA signaling. Furthermore, the action mode of bestatin also supports the prediction that a mutant plant harboring mutation of bestatin target will be insensitive to bestatin but hypersensitive to JA; this phenotype exactly fits the phenotype of our second group of ber mutants (i.e. JA-hypersensitive ber mutants) that includes ber157, ber544, and ber426. Our ongoing efforts to characterize these mutants, identify the corresponding genes, and perform functional analyses will be of great importance for the elucidation of the molecular mechanisms of bestatin biological function and further understanding of the molecular basis of JA signaling.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
LITERATURE CITED
 

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., 2003Go) and jai1-1 (Li et al., 2004Go) were in the genetic background of cv Castlemart. Tomato plants were grown at standard conditions as described previously (Schaller et al., 1995Go; Li et al., 2003Go). 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 µL 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, 1967Go). 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 (µg/mL leaf juice) were determined using the following formula: (diameter x diameter – 625) x 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 0.5x 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 0.5x 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., 2005Go). Ten micrograms of total RNA was separated in an agarose gel containing 10% formaldehyde, blotted onto a Hybond N+ 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'-TTGCCTTGGGTTCATCACTCT-3') for PI-II from tomato, and AtVSP1F (5'-ATGAAAATCCTCTCACTTTCA-3') and AtVSP1R (5'-TATCCATATTTAGCGTAGTAGG-3') for AtVSP1 from Arabidopsis (Col-0).


Plasmid Construction and Plant Transformation

A 740-bp fragment upstream of the AtVSP1 coding sequence was isolated by PCR from genomic DNA (Col-0) using the primers 5'-AAGAAAATCAAGCTTTAACCTAAAATCAAC-3' and 5'-GTCGGATCCAGTTTATGGTGTTTATTTGTG-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), which was used for transformation of Arabidopsis by vacuum infiltration (Bechtold et al., 1993Go). A homozygous transgenic line of AtVSP1::GUS was identified and used for GUS staining experiment.


Analysis of GUS Activity

Histochemical staining for GUS activity in transgenic plants was performed as described previously (Jefferson et al., 1987Go).


Microarray Experiments and Data Analysis

Two-week-old, light-grown Arabidopsis seedlings were sprayed with 50 µM bestatin or 50 µM MeJA 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 RNA preparation used as control. Total RNA was 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-sided Wilcoxon'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 compute 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 –1.2 (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 0.5x MS medium containing 25 µM bestatin under white light (16-h-light/8-h-dark cycle) at 22°C for 8 to 10 d. Seedlings with elongated roots were selected as putative ber mutants and directly transplanted to soil. Resulting M3 seeds from the putative mutants were retested on 25 µM 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 backcrossed at least twice to wild type before physiological and molecular 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 analysis using the standard procedures as described (Lukowitz et al., 2000Go).


   ACKNOWLEDGMENTS
 
We gratefully acknowledge Dr. Clarence Ryan (Washington State University, 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. Salomé 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; returned for revision June 10, 2006; accepted June 11, 2006.


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

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Chuanyou Li (cyli{at}genetics.ac.cn).

[W] The online version of this article contains Web-only data. Back

[OA] Open Access articles can be viewed online without a subscription. Back

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.106.080390.

* Corresponding author; e-mail cyli{at}genetics.ac.cn; fax 8610–6487–3428.


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MYC2 Differentially Modulates Diverse Jasmonate-Dependent Functions in Arabidopsis
PLANT CELL, July 1, 2007; 19(7): 2225 - 2245.
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T. A. Walsh, T. Bauer, R. Neal, A. O. Merlo, P. R. Schmitzer, G. R. Hicks, M. Honma, W. Matsumura, K. Wolff, and J. P. Davies
Chemical Genetic Identification of Glutamine Phosphoribosylpyrophosphate Amidotransferase as the Target for a Novel Bleaching Herbicide in Arabidopsis
Plant Physiology, July 1, 2007; 144(3): 1292 - 1304.
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