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. 2009 Nov;151(3):1412–1420. doi: 10.1104/pp.109.140202

A Leaky Mutation in DWARF4 Reveals an Antagonistic Role of Brassinosteroid in the Inhibition of Root Growth by Jasmonate in Arabidopsis1,[C]

Chunmei Ren 1, Chengyun Han 1, Wen Peng 1, Ying Huang 1, Zhihong Peng 1, Xingyao Xiong 1, Qi Zhu 1, Bida Gao 1, Daoxin Xie 1,*
PMCID: PMC2773060  PMID: 19741050

Abstract

The F-box protein CORONATINE INSENSITIVE1 (COI1) plays a central role in jasmonate (JA) signaling and is required for all JA responses in Arabidopsis (Arabidopsis thaliana). To dissect JA signal transduction, we isolated the partially suppressing coi1 (psc1) mutant, which partially suppressed coi1 insensitivity to JA inhibition of root growth. The psc1 mutant partially restored JA sensitivity in coi1-2 background and displayed JA hypersensitivity in wild-type COI1 background. Genetic mapping, sequence analysis, and complementation tests revealed that psc1 is a leaky mutation of DWARF4 (DWF4) that encodes a key enzyme in brassinosteroid (BR) biosynthesis. Physiological analysis showed that an application of exogenous BR eliminated the partial restoration of JA sensitivity by psc1 in coi1-2 background and the JA hypersensitivity of psc1 in wild-type COI1 background. Exogenous BR also attenuated JA inhibition of root growth in the wild type. In addition, the expression of DWF4 was inhibited by JA, and this inhibition was dependent on COI1. These results indicate that (1) BR is involved in JA signaling and negatively regulates JA inhibition of root growth, and (2) the DWF4 is down-regulated by JA and is located downstream of COI1 in the JA-signaling pathway.

The plant hormone jasmonates, which include jasmonic acid and its cyclopentanone derivatives as well as cyclopentenones, regulate a variety of plant developmental processes including root growth, pollen development, senescence, and trichome development (McConn and Browse, 1996; Li et al., 2004; Browse, 2005; Schilmiller et al., 2007; Shan et al., 2007; Wasternack, 2007; Yan et al., 2007; Balbi and Devoto, 2008). Jasmonates also mediate responses to stress, wounding, insect attack, pathogen infection, and UV damage (Reymond and Farmer, 1998; Reymond et al., 2000, 2004; Bodenhausen and Reymond, 2007; Browse and Howe, 2008; Howe and Jander, 2008; Farmer and Dubugnon, 2009).

The effects of jasmonate (JA) on Arabidopsis (Arabidopsis thaliana) have been defined mainly through genetic analysis of JA biosynthetic mutants such as fad3/fad7/fad8 (McConn and Browse, 1996), opr3/dde1 (Sanders et al., 2000; Stintzi and Browse, 2000), and aos (Park et al., 2002), and through genetic analysis of JA-signaling mutants including jar1 (Staswick et al., 1992), coronatine insensitive1-1 (coi1-1; Feys et al., 1994), jin1, and jin4 (Berger et al., 1996). Among these mutants, coi1-1 is completely deficient in all the JA responses (Feys et al., 1994; Xie et al., 1998; Reymond et al., 2000; Browse, 2009; Shan et al., 2009; Sun et al., 2009). The coi1-1 mutant has defects in JA-inhibited root growth, JA-induced anthocyanin accumulation, JA-induced lateral root formation, and JA-regulated gene expression, exhibits male sterility, and is susceptible to insect attack and pathogen infection, thereby having been considered as a key regulator in the JA signal transduction pathway.

The COI1 gene has been found to encode an F-box protein, providing the first indication that ubiquitin-mediated protein degradation is involved in JA signaling (Xie, et al., 1998). This hypothesis has been supported by the demonstration that COI1 interacts with Arabidopsis CULLIN1, RBX1, and Skp1-like proteins ASK1 or ASK2 (Liu et al., 2004) to assemble SCFCOI1 complexes in planta (Xu et al., 2002; Wang et al., 2005), and by observations that mutations in genes required for SCF function, such as AXR1 and CULLIN1, result in reduced JA responses (Tiryaki and Staswick, 2002; Ren et al., 2005). JAZ proteins were identified as the substrates of the SCFCOI1 complex for degradation by the 26S proteasome in response to JA (Chini et al., 2007; Thines et al., 2007; Chico et al., 2008; Katsir et al., 2008a). The complex containing COI1 and JAZ proteins might be a reception site of the jasmonoyl Ile (Katsir et al., 2008b), which is active as a specific enantiomeric form, the (+)-7-iso-jasmonoyl-l-Ile (Fonseca et al., 2009). Recent study demonstrated that COI1 is a JA receptor (Yan et al., 2009). Together, these research findings have gradually uncovered the molecular mechanism for the role of COI1 in JA signaling.

To understand the molecular mechanism by which COI1 regulates JA responses, we previously conducted a genetic screen for suppressors of the coi1 mutant and identified cos1 as a suppressor of coi1 (Xiao et al., 2004). To further investigate COI1-mediated JA responses and dissect JA-signaling pathway, we continued to carry out genetic screens for suppressors of coi1 mutant, and isolated one mutant named partially suppressing coi1 (psc1) insensitivity to JA. Genetic mapping and a complementation test revealed that PSC1 is an allele of DWARF4 (DWF4) that encodes a key enzyme in brassinosteroid (BR) biosynthesis (Choe et al., 1998). Physiological analysis showed that the psc1 mutation partially restored JA inhibition of root growth in coi1-2 background and displayed JA hypersensitivity in wild-type COI1 background; the effects of psc1 were eliminated by exogenous BR. Furthermore, we found that BR repressed JA sensitivity in wild-type seedlings and that the inhibition of DWF4 expression by JA was dependent on COI1. These results indicate that BR is involved in JA signaling and negatively regulates JA inhibition of root growth, and that DWF4 is down-regulated by JA and is located downstream of COI1 in the JA-signaling pathway.

RESULTS

Isolation of the coi1 Suppressors

To isolate mutants that suppress coi1, we screened approximately 100,000 M2 seedlings from approximately 20,000 M1 ethyl methanesulfonate-mutagenized seeds of coi1-2, a coi1 leaky mutant resistant to JA, but partially fertile and able to produce a small quantity of seeds (Xu et al., 2002), for reduced resistance to JA. Suppressor candidates of coi1 were selected based on seedling phenotype with shorter roots and stunted growth when grown on a medium containing 10 μm methyl JA (MeJA). One suppressor candidate exhibited partial but obvious root growth inhibition by 10 μm MeJA, which was determined to have a single recessive Mendelian locus and named psc1. This psc1coi1 mutant (homozygous for both psc1 and coi1-2 mutations) was backcrossed to coi1-2 four times to remove other potential mutations. Further analysis of the inhibition of root growth by JA showed that relative root elongation of psc1coi1 was clearly less than that of coi1-2, though it was still higher than that of wild type (Fig. 1A). Approximately 9%, 30%, and 54% inhibition of root elongation by 10 μm MeJA was observed in coi1-2, psc1coi1, and the wild type, respectively (Fig. 1A). These results indicated that the psc1 mutation partially restores the JA sensitivity in the coi1 mutant background.

Figure 1.

Figure 1.

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psc1coi1 exhibited increased JA inhibition of root growth and morphologic alteration. A, Effect of MeJA on root growth of psc1coi1. Five-day-old seedlings transferred from MS to MS medium containing 0, 1, 5, and 10 μm MeJA were grown on vertically oriented plates for 3 d and increase in root length was measured. Relative root elongation is expressed as a percentage of root elongation on MS medium. Error bars represent se (n > 30). B, The morphology of wild-type (WT), coi1-2, and psc1coi1 seedlings at 18 d (top section) and flowering plants at 6 weeks (bottom section).

In addition to the partial root growth inhibition by JA, the psc1coi1 exhibited other alterations in plant morphology. For example, the rosette leaves were smaller and round, the petioles were shorter, and the plant height was reduced significantly compared to coi1-2 (Fig. 1B).

Map-Based Cloning of the PSC1 Gene

To map the PSC1 locus, we carried out the genetic crossing between psc1coi1, which is of the Columbia (Col) ecotype, and coi1-12, a Landsberg erecta JA-insensitive mutant containing a single amino acid replacement (from Phe 359 to Lys) in COI1 (Xiao et al., 2004). We subsequently screened the F2 progeny for psc1coi1 homozygous seedlings that exhibited partial root growth inhibition by JA.

Based on the linkage analysis among molecular markers and the psc1 phenotype of about 2,000 psc1coi1 seedlings (about 25% of the F2 population), we mapped the PSC1 gene onto an approximately 45-kb interval between simple sequence length polymorphic (SSLP) marker S18800 and cleaved-amplified polymorphic sequence (CAPS) marker C18845, which were localized onto the T3A5 bacterial artificial chromosome (BAC; Fig. 2A). Sequencing verification identified a mutation within the DWF4 gene: A single cytosine nucleotide at the 1,403rd base in the coding region of the DWF4 gene was replaced by a thymine nucleotide, resulting in the substitution of the 468th amino acid in the DWF4 protein, Ala, with Val (Fig. 2B).

Figure 2.

Figure 2.

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Mapping of the PSC1 gene. A, With the SSLP markers S17854 and CIW4, CAPS marker MS-3-1, SSLP marker S18800, and CAPS marker C18845 in order, the PSC1 locus was mapped to a approximately 45-kb interval between S18800 and C18845 on chromosome 3. The BAC T3A5 covers the PSC1 locus region, and its two conjoint BACs, T20E23 and F18B3, are shown. The positions below the markers indicate the locations of the Arabidopsis Genome Initiative map on the chromosome. The numbers in parentheses indicate the recombinants. B, Sequence analysis of the DWF4 gene in the psc1coi1 mutant has a one-base change from C to T at the 1,403rd base, resulting in substitution of Val for Ala. [See online article for color version of this figure.]

We transferred a genomic fragment containing wild-type DWF4 gene with its endogenous promoter into the psc1coi1 mutant and generated psc1coi1∷tDWF4 transgenic plants (see “Materials and Methods”) for genetic complementation test. As shown in Figure 3A, the psc1coi1∷tDWF4 seedling was similar to the coi1-2 seedling. When seedlings were grown on Murashige and Skoog (MS) supplemented with various concentrations of MeJA, relative root elongation of psc1coi1∷tDWF4 showed an obvious increase compared with that of psc1coi1, and was comparable to that of coi1-2 (Fig. 3B). Also, other phenotypes including the size and status of leaves as well as height of plants were all restored to those of coi1-2 (Fig. 3C). These results demonstrated that PSC1 is an allele of DWF4 and that the psc1 mutation in the psc1coi1 mutant is responsible for the partial suppression on coil insensitivity to JA-inhibitory root growth.

Figure 3.

Figure 3.

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DWF4 restores both JA insensitivity and morphologic phenotype in psc1coi1. A, The phenotype of 12-d-old seedlings grown on MS medium with 10 μm MeJA. B, Effect of MeJA on root growth of coi1-2, psc1coi1, and transgenic psc1coi1∷tDWF4 seedlings. Five-day-old seedlings transferred from MS to MS medium containing 0, 1, 5, and 10 μm MeJA were grown on vertically oriented plates for 3 d and increase in root length was measured. Relative root elongation is expressed as a percentage of root elongation on MS medium. Error bars represent se (n > 30). C, The morphology of 18-d-old seedlings (top section) and 8-week-old flowering plants (bottom section).

Cross Talk between JA and BR Signaling Pathways

Because the DWF4 gene encodes a key enzyme in BR biosynthesis (Choe et al., 1998) and the mutants in BR biosynthesis are dwarf plants with short roots (Azpiroz et al., 1998; Nemhauser and Chory, 2004), we first investigated whether the short root of psc1coi1 seedlings could be rescued by an application of exogenous BR, epibrassinolide (epi-BL; the most active BR). As expected, root elongation of psc1coi1 was less than that of wild type or coi1-2 when seedlings were grown on MS medium (Fig. 4A). When treated with 10 nm epi-BL, root elongation of psc1coi1 appeared similar to that of wild type or coi1-2 (Fig. 4A). Treatment with 10 nm epi-BL was sufficient to rescue the short root of psc1coi1 seedlings and had no obvious effect on the root growth of the wild-type or coi1-2 seedlings, but higher concentrations of epi-BL (100 or 1,000 nm) inhibited root growth (Fig. 4A). These results suggest that the mutation of DWF4 in psc1coi1 leads to a defect in BR biosynthesis.

Figure 4.

Figure 4.

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BR eliminates the effect of psc1 on the sensitivity of coi1 to JA and attenuates JA sensitivity in the wild type. A, Effect of epi-BL (the most active BR) on root growth of wild-type (WT), coi1-2, and psc1coi1 seedlings. Five-day-old seedlings transferred to MS medium with 0, 0.1, 1, 10, 100, and 1,000 nm epi-BL were grown on vertically oriented plates for 3 d and increase in root length was measured. Error bars represent se (n > 30). B, Effect of MeJA and epi-BL on root growth of coi1-2 and psc1coi1 seedlings. Five-day-old seedlings transferred to MS medium containing 0, 5, and 10 μm MeJA with or without 10 nm epi-BL were grown on vertically oriented plates for 3 d and increase in root length was measured. Relative root elongation is expressed as a percentage of root elongation on MS with (right section) or without (left section) 10 nm epi-BL. Error bars represent se (n > 30). C, Effect of MeJA and epi-BL on root growth of wild-type seedlings. Five-day-old seedlings transferred to MS medium containing 0, 5, 10, and 25 μm MeJA with or without 10 nm epi-BL were grown on vertically oriented plates for 3 d and increase in root length was measured. Error bars represent se (n > 30).

The psc1 mutation affected BR biosynthesis and caused JA partial sensitivity in coi1-2 background, implying a cross talk between JA and BR. To verify the cross talk between JA and BR, we tested whether the suppression of coi1 by psc1 could be eliminated by 10 nm epi-BL, a concentration of epi-BL that did not affect the root growth of the wild type or coi1-2 but rescued the root growth of psc1coi1 to the normal wild-type level (Fig. 4A). As shown in Figure 4B, the inhibition of root growth by JA in psc1coi1 seedlings was reduced remarkably by epi-BL and was restored to that of coi1-2. Therefore, BR is able to eliminate the suppression of psc1 on coil insensitivity to JA-inhibitory root growth, suggesting that the BR signal might negatively regulate JA inhibition of root growth.

To further determine whether the BR signal negatively regulates JA inhibition of root growth, we investigated whether BR was able to attenuate JA inhibition of root growth in the wild type. The wild-type seedlings were grown on MS medium supplemented with 5, 10, and 25 μm MeJA with or without 10 nm epi-BL. As shown in Figure 4C, JA inhibition of root growth in the wild-type seedlings was partially attenuated by epi-BL. All seedlings treated with epi-BL exhibited less sensitivity to JA, suggesting that the BR signal partially counters JA inhibition of root growth.

The psc1 Single Mutant Exhibits JA Hypersensitivity

We generated the psc1 single mutant in wild-type COI1 background (see “Materials and Methods”) and found that the morphologic phenotypes of the psc1 single mutant, such as size of seedlings, rosette leaves, and plant height, were similar to those of the psc1coi1 double mutant except that fertility was almost normal in the psc1 single mutant but partial in psc1coi1 (Figs. 1B, 3C, and 5A). The transgenic plant (psc1∷tDWF4) expressing wild-type DWF4 with its endogenous promoter in psc1 (see “Materials and Methods”) displayed wild-type-like morphologic phenotypes (Fig. 5A). As shown in Figure 5B, root elongation of seedlings treated with 10 nm epi-BL was similar for psc1 and the wild type. These results suggest that the wild-type DWF4 gene complements the phenotypes in the psc1 mutant and that the application of exogenous BR is able to rescue the root growth of the psc1 mutant.

Figure 5.

Figure 5.

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Phenotypes of the psc1 single mutant. A, The morphology of wild-type (WT), psc1, and transgenic psc1∷tDWF4 seedlings at 21 d (top section) and flowering plants at 6 weeks (bottom section). B, Effect of epi-BL (the most active BR) on root growth of wild-type (WT) and psc1 seedlings. Five-day-old seedlings transferred to MS medium with 0, 0.1, 1, 10, 100, and 1,000 nm epi-BL were grown on vertically oriented plates for 3 d and increase in root length was measured. Error bars represent se (n > 30).

Relative root elongation was less for psc1 than for the wild type when seedlings were grown on a medium with JA (left section of Fig. 6). Exposure to 10 μm MeJA reduced root length by 70% in the psc1 mutant but by only 54% in the wild type, demonstrating that the psc1 mutant deficient in BR biosynthesis was more sensitive to JA than the wild type.

Figure 6.

Figure 6.

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BR eliminates JA hypersensitivity in the psc1 single mutant. Five-day-old seedlings transferred to MS medium containing 0, 5, and 10 μm MeJA with or without 10 nm epi-BL were grown on vertically oriented plates for 3 d and increase in root length was measured. Relative root elongation is expressed as a percentage of root elongation on MS medium with (right section) or without (left section) 10 nm epi-BL. Error bars represent se (n > 30). WT, Wild type.

To test whether the JA hypersensitivity in the psc1 single mutant could be eliminated by exogenous BR, the seedlings were grown on MS medium supplemented with various concentrations of MeJA and without or with 10 nm epi-BL. As shown in right section of Figure 6, relative root elongation in the presence of epi-BL was similar for psc1 and the wild type, indicating that BR can completely depress JA hypersensitivity in the psc1 single mutant. These results further confirm that the defect of BR biosynthesis in psc1 increases sensitivity to JA.

JA Inhibits DWF4 Expression in COI1-Dependent Manner

We next investigated whether JA affects the expression of DWF4. Because DWF4 transcripts were rarely detected by northern blotting (Kim et al., 2006; data not shown), reverse transcription (RT)-PCR was used to analyze the expression of the DWF4 gene. As shown in Figure 7, the amplified transcripts of DWF4 observably decreased upon JA treatment in wild type, suggesting that JA inhibits the expression of DWF4. To determine whether the inhibition on the DWF4 expression by JA is dependent on COI1, we treated the null mutant coi1-1 plants with JA for various periods. We found that level of DWF4 expression was not significantly altered in coi1-1 treated with or without JA (Fig. 7). These data demonstrate that JA inhibits DWF4 expression in COI1-dependent manner.

Figure 7.

Figure 7.

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Analysis of DWF4 expression by RT-PCR. The total RNA was extracted from rosette leaves of 4-week-old plants with treatment of 100 μm MeJA for 8, 24, and 48 h, or water for 8 h (CK), and then was used in RT-PCR to examine the expression of DWF4. The ACT1 fragment was amplified as a control. WT, Wild type, Col-0.

DISCUSSION

By using genetic screens for suppressors, we isolated the psc1 mutant that partially suppresses the JA insensitivity of coi1 (Fig. 1A). Genetic mapping and a complementation test revealed that PSC1 is an allele of the DWF4 gene, in which the 468th amino acid has changed from Ala to Val (Fig. 2B). The DWF4 gene encodes a cytochrome P450 that mediates multiple 22α-hydroxylation steps in BR biosynthesis (Choe et al., 1998).

BR is a family of polyhydroxylated steroid hormones involved in many aspects of plant growth and development (Belkhadir and Chory, 2006; Wang et al., 2006; Gendron et al., 2008; Tang et al., 2008). The growth of mutants defective in BR biosynthesis and signaling is severely retarded (Azpiroz et al., 1998; He et al., 2005; Belkhadir and Chory, 2006). Compared with dwf4-102 (Nakamoto, et al., 2006), a null mutant of DWF4 that is plant lethal (Nakamoto et al., 2006; data not shown), the psc1 mutant showed a dwarf phenotype that included shorter petioles, round and smaller rosette leaves, and reduced plant height (Fig. 5A), but the fertility of the psc1 single mutant was almost normal (Fig. 5A), indicating that psc1 is a leaky mutation in DWF4.

Physiological analysis of roots revealed that psc1 partially restored JA sensitivity in coi1-2 background (Fig. 1A) and exhibited JA hypersensitivity in wild-type COI1 background (Fig. 6). Both of these responses to JA were eliminated by exogenous BR (Figs. 4B and 6), whereas exogenous BR attenuated JA sensitivity in the wild type (Fig. 4C), suggesting that BR is involved in JA signaling and negatively regulates JA inhibition of root growth.

Upon BR treatment, several BR synthesis genes, including DWF4 and CPD encoding C-23 hydroxylase (Szekeres et al., 1996), were down-regulated and a BR inactivation gene (BAS1) was up-regulated (Tanaka et al., 2005; Kim et al., 2006). However, when BR was depleted by treatment with brassinazole, a BR biosynthesis inhibitor, the expression of several BR synthesis genes, including DWF4, CPD, and DET2, increased (Tanaka et al., 2005). In this study, we found that JA inhibited the expression of DWF4 (Fig. 7), which was consistent with the result generated by Genevestigator (Zimmermann et al., 2004; http://www.arabidopsis.org). Furthermore, the data from Genevestigator expression analysis (http://www.arabidopsis.org; Zimmermann et al., 2004) showed that MeJA treatment reduced the expression of CPD and BAS1 but induced expression of DET2. In the BR-biosynthesis pathway, DWF4 locates downstream of DET2 and upstream of CPD and catalyzes the rate-limiting step (Choe et al., 1998). Therefore, we hypothesized that JA treatment might reduce endogenous BR by regulation of the expression of BR biosynthetic genes including DWF4.

Taken together, we proposed a model for psc1 to exhibit a partial restoration of JA-inhibitory root growth in the coi1-2 background and JA hypersensitivity in the COI1 wild-type background. BR signal negatively regulates JA-inhibitory root growth. Reduction of BR synthesis would reduce the negative effect of BR signal on JA-inhibitory root growth and enhance JA sensitivity of root growth. The psc1 mutation (a leaky mutation of DWF4) partially reduces normal BR synthesis, whereas partial reduction of BR synthesis in psc1 would partially reduce the negative effect of BR signal on JA-inhibitory root growth. As a result, psc1 shows an increased JA sensitivity of root growth in both coi1-2 and wild-type background. The psc1coi1 mutant exhibited partial sensitivity to JA-inhibitory root growth compared with the coi1-2 mutant that is resistant to JA, and the psc1 mutant is more sensitive to JA-inhibitory root growth compared with the wild type.

Generally speaking, JA inhibits plant root growth and also induces expression of many genes including VSP1, LOX2, Thi2.1, and JAZs (Xu et al., 2002; Chini et al., 2007; Thines et al., 2007; Yan et al., 2007; Chico et al., 2008; Katsir et al., 2008a), and regulates anthocyanin accumulation (Shan et al., 2009) and lateral root formation (Sun et al., 2009). However, we found that the psc1 mutation, which partially increased sensitivity of JA-inhibitory root growth, failed to restore expression of VSP1, LOX2, Thi2.1, and JAZ9 in coi1-2 background (data not shown). In contrast, the expression of JA-induced gene VSP1 appeared to decrease in psc1coi1 compared with coi1-2, and VSP1 expression was also reduced in the psc1 single mutant compared with wild type (data not shown). Consistent with this observation, the data from Genevestigator expression analysis (http://www.arabidopsis.org; Zimmermann, et al., 2004) showed that BR treatment increased the expression of some JA-inducible genes including LOX2, Thi2.1, and JAZ9. Similarly, we also found that the psc1 mutation failed to restore JA-induced anthocyanin accumulation or JA-induced lateral root formation in coi1-2 background (data not shown). The cross talk between JA and BR could be very complicated, as is the case for the cross talk between JA and ethylene where these two hormones can either work cooperatively or antagonistically in the regulation of different stress responses and developmental processes (Lorenzo and Solano, 2005).

MATERIALS AND METHODS

Plant Materials and Growth Conditions

The coi1-2 leaky mutant was identified previously in our laboratory (Xu et al., 2002).

Seeds were surfaced sterilized, plated on plant growth medium (MS supplemented with 1% Suc; Sigma), chilled at 4°C for 3 d, and then transferred to a growth room with a 16-h-light (22°C–24°C)/8-h-dark (16°C–19°C) photoperiod.

Mutant Screening

Approximately 30,000 seeds of Arabidopsis (Arabidopsis thaliana) coi1-2 were mutagenized with 0.3% ethyl methanesulfonate following routine procedures. About 70% of the mutagenized seeds (referred to as the M1 population) could grow in soil and generate M2 seeds. M2 seeds were routinely plated on MS medium containing 10 μm MeJA (Aldrich) to screen for mutants sensitive to MeJA, i.e. mutants with shorter roots and stunted growth relative to coi1-2.

Generation of the psc1 Single Mutant

The psc1coi1 mutant was crossed to the wild type, Col-0, and then the plants (homozygote of psc1 and heterozygote or homozygote of COI1) were selected from the F2 population based on their increased sensitivity to MeJA (relative to psc1coi1) and dwarf phenotype. The psc1 single mutant was then identified by sequencing both COI1 and PSC1.

Measurement of Root Elongation

Seedlings were grown on MS medium for 5 d and then transferred to MS medium supplemented with various concentrations of MeJA and/or epi-BL (Sigma). The position of the root tips was marked, and plates were placed vertically in the growth room. Three days later, increase in root length was measured for more than 30 seedlings. All experiments were repeated three to five times.

Molecular Markers

The CAPS markers MS_3_1 and C18845 show a DNA polymorphism between Col and Landsberg erecta when HincII (for MS_3_1) and Bsp1407I (for C18845) were used to digest the PCR fragment amplified with their corresponding primers (MS_3_1, 5′-GAGAGTAAACTTGACAATTACAAGAGA-3′ and 5′-TTCCCAATTTTTTCCAAGTTTTTAGGG-3′; C18845, 5′-ACGCATTTAGCACTCTGATG-3′ and 5′-TGTCAGCTTCTATTGGATTG-3′). The SSLP markers S17854, S18800, and CIW4 show a polymorphism of difference in size of 13, 16, and 25 bp, respectively, between Col and Landsberg erecta when the PCR fragment was amplified with their corresponding primers (S17854, 5′-AACATGGTAAAGCCAAAATCA-3′ and 5′-AATGCATTAGACGAATGATTCA-3′; S18800, 5′-GGAAAAGCCAGCCAATTATA-3′ and 5′-CAGTGCAATTAGTGCATATC-3′; CIW4, 5′-GTTCATTAAACTTGCGTGTGT-3′ and 5′-TACGGTCAGATTGAGTGATTC-3′).

Complementation Test

A 5,122-bp genomic fragment (referred to as tDWF4) containing the DWF4 promoter region and the coding sequence was amplified by Pfu DNA polymerase (Stratagene) from the wild type (Col-0) using forward primer P1 (5′-ACTTGAGCTCAAACATTACGGGACACTGGACTC-3′) and reverse primer P2 (5′-AAAACCCGGGCAGAATACGAGAAACCCTAATA-3′). The amplified fragment was cloned into pFlag (Ren et al., 2005) at the SacI/SmaI sites.

The construct was verified by sequencing and then introduced into psc1coi1 by the floral-dip method of in planta Agrobacterium tumefaciens-mediated transformation (Clough and Bent, 1998). Three independent lines were analyzed in detail and exhibited similar phenotypes to each other. Data from one of lines, psc1coi1∷tDWF4, were representative and are shown in the figures. The psc1∷tDWF4 line was generated from the cross of psc1coi1∷tDWF4 with psc1 and was screened for presence of the tDWF4 transgene and the psc1 mutation but absence of coi1-2.

RT-PCR Analysis

The rosette leaves of 4-week-old plants grown in soil were drenched in 100 μm MeJA for 8, 24, and 48 h, or in water for 8 h (control), and then harvested. RT-PCR analysis was performed following routine procedures. The DWF4 gene was amplified with primers 5′-GGTCGATGCTTGTTCTTGTTGGT-3′ and 5′-GCTCCGTTGTTTTGCTGTTGC-3′, and the ACT1 gene was amplified with primers 5′-TGGGTCGTCCTCGTCACA-3′ and 5′-GATACCAGCATTCTCCATACCA-3′. The PCR program consisted of an initial denaturing at 95°C for 2 min; followed by 26 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 60 s; and a final extension at 72°C for 10 min.

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession number AY090266.

Acknowledgments

We thank Dr. Guo-Liang Wang for useful suggestions.

1

This work was supported by the National Science Foundation of China, the National Basic Research 973 Program of China, and the Scientific Research Fund of Hunan Provincial Education Department.

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: Daoxin Xie (daoxinlab@tsinghua.edu.cn).

[C]

Some figures in this article are displayed in color online but in black and white in the print edition.

References

  1. Azpiroz R, Wu Y, LoCascio JC, Feldmann KA (1998) An Arabidopsis brassinosteroid-dependent mutant is blocked in cell elongation. Plant Cell 10 219–230 [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Balbi V, Devoto A (2008) Jasmonate signalling network in Arabidopsis thaliana: crucial regulatory nodes and new physiological scenarios. New Phytol 177 301–318 [DOI] [PubMed] [Google Scholar]
  3. Belkhadir Y, Chory J (2006) Brassinosteroid signaling: a paradigm for steroid hormone signaling from the cell surface. Science 314 1410–1411 [DOI] [PubMed] [Google Scholar]
  4. Berger S, Bell E, Mullet JE (1996) Two methyl jasmonate insensitive mutants show altered expression of AtVsp in response to methyl jasmonate and wounding. Plant Physiol 111 525–531 [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bodenhausen N, Reymond P (2007) Signaling pathways controlling induced resistance to insect herbivores in Arabidopsis. Mol Plant Microbe Interact 20 1406–1420 [DOI] [PubMed] [Google Scholar]
  6. Browse J (2005) Jasmonate: an oxylipin signal with many roles in plants. Vitam Horm 72 431–456 [DOI] [PubMed] [Google Scholar]
  7. Browse J (2009) Jasmonate passes muster: a receptor and targets for the defense hormone. Annu Rev Plant Biol 60 183–205 [DOI] [PubMed] [Google Scholar]
  8. Browse J, Howe GA (2008) New weapons and a rapid response against insect attack. Plant Physiol 146 832–838 [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chico JM, Chini A, Fonseca S, Solano R (2008) JAZ repressors set the rhythm in jasmonate signaling. Curr Opin Plant Biol 11 486–494 [DOI] [PubMed] [Google Scholar]
  10. Chini A, Fonseca S, Fernandez G, Adie B, Chico JM, Lorenzo O, Garcia-Casado G, Lopez-Vidriero I, Lozano FM, Ponce MR, et al (2007) The JAZ family of repressors is the missing link in jasmonate signalling. Nature 448 666–671 [DOI] [PubMed] [Google Scholar]
  11. Choe S, Dilkes BP, Fujioka S, Takatsuto S, Sakurai A, Feldmann KA (1998) The DWF4 gene of Arabidopsis encodes a cytochrome P450 that mediates multiple 22a-hydroxylation steps in brassinosteroid biosynthesis. Plant Cell 10 231–243 [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16 735–743 [DOI] [PubMed] [Google Scholar]
  13. Farmer EE, Dubugnon L (2009) Detritivorous crustaceans become herbivores on jasmonate-deficient plants. Proc Natl Acad Sci USA 106 935–940 [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Feys BJF, Benedetti CE, Penfold CN, Turner JG (1994) Arabidopsis mutants selected for resistance to the phytotoxin coronatine are male-sterile, insensitive to methyl jasmonate, and resistant to a bacterial pathogen. Plant Cell 6 751–759 [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Fonseca S, Chini A, Hamberg M, Adie B, Porzel A, Kramell R, Miersch O, Wasternack C, Solano R (2009) (+)-7-iso-jasmonoyl-L-isoleucine is the endogenous bioactive jasmonate. Nat Chem Biol 5 344–350 [DOI] [PubMed] [Google Scholar]
  16. Gendron JM, Haque A, Gendron N, Chang T, Asami T, Wang ZY (2008) Chemical genetic dissection of brassinosteroid-ethylene interaction. Mol Plant 1 368–379 [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. He JX, Gendron JM, Sun Y, Gampala SS, Gendron N, Sun CQ, Wang ZY (2005) BZR1 is a transcriptional repressor with dual roles in brassinosteroid homeostasis and growth responses. Science 307 1634–1638 [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Howe GA, Jander G (2008) Plant immunity to insect herbivores. Annu Rev Plant Biol 59 41–66 [DOI] [PubMed] [Google Scholar]
  19. Katsir L, Chung HS, Koo AJK, Howe GA (2008. a) Jasmonate signaling: a conserved mechanism of hormone sensing. Curr Opin Plant Biol 11 428–435 [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Katsir L, Schilmiller AL, Staswick PE, He SY, Howe GA (2008. b) COI1 is a critical component of a receptor for jasmonate and the bacterial virulence factor coronatine. Proc Natl Acad Sci USA 105 7100–7105 [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kim HB, Kwon M, Ryu H, Fujioka S, Takatsuto S, Yoshida S, An CS, Lee I, Hwang I, Choe S (2006) The regulation of DWARF4 expression is likely a critical mechanism in maintaining the homeostasis of bioactive brassinosteroids in Arabidopsis. Plant Physiol 140 548–557 [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Li L, Zhao Y, McCaig BC, Wingerd BA, Wang J, Whalon ME, Pichersky E, Howe GA (2004) The tomato homolog of CORONATINE-INSENSITIVE1 is required for the maternal control of seed maturation, jasmonate-signaled defense responses, and glandular trichome development. Plant Cell 16 126–143 [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Liu F, Ni W, Griffith M, Huang Z, Chang C, Peng W, Ma H, Xie D (2004) The ASK1 and ASK2 genes are essential for Arabidopsis early development. Plant Cell 16 5–20 [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lorenzo O, Solano R (2005) Molecular players regulating the jasmonate signalling network. Curr Opin Plant Biol 8 532–540 [DOI] [PubMed] [Google Scholar]
  25. McConn M, Browse J (1996) The critical requirement for linolenic acid is pollen development, not photosynthesis, in an Arabidopsis mutant. Plant Cell 8 403–416 [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Nakamoto D, Ikeura A, Asami T, Yamamoto KT (2006) Inhibition of brassinosteroid biosynthesis by either a dwarf4 mutation or a brassinosteroid biosynthesis inhibitor rescues defects in tropic responses of hypocotyls in the Arabidopsis mutant nonphototropic hypocotyl 4. Plant Physiol 141 456–464 [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Nemhauser JL, Chory J (2004) BRing it on: new insights into the mechanism of brassinosteroid action. J Exp Bot 55 265–270 [DOI] [PubMed] [Google Scholar]
  28. Park JH, Halitschke R, Kim HB, Baldwin IT, Feldmann KA, Feyereisen R (2002) A knock-out mutation in allene oxide synthase results in male sterility and defective wound signal transduction in Arabidopsis due to a block in jasmonic acid biosynthesis. Plant J 31 1–12 [DOI] [PubMed] [Google Scholar]
  29. Ren C, Pan J, Peng W, Genschik P, Hobbie L, Hellmann H, Estelle M, Gao B, Peng J, Sun C, et al (2005) Point mutations in Arabidopsis Cullin1 reveal its essential role in jasmonate response. Plant J 42 514–524 [DOI] [PubMed] [Google Scholar]
  30. Reymond P, Bodenhausen N, Van Poecke RMP, Krishnamurthy V, Dicke M, Farmer EE (2004) A conserved transcript pattern in response to a specialist and a generalist herbivore. Plant Cell 16 3132–3147 [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Reymond P, Farmer EE (1998) Jasmonate and salicylate as global signals for defense gene expression. Curr Opin Plant Biol 1 404–411 [DOI] [PubMed] [Google Scholar]
  32. Reymond P, Weber H, Damond M, Farmer EE (2000) Differential gene expression in response to mechanical wounding and insect feeding in Arabidopsis. Plant Cell 12 707–719 [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Sanders PM, Lee PY, Biesgen C, Boone JD, Beals TP, Weiler EW, Goldberg RB (2000) The Arabidopsis DELAYED DEHISCENCE1 gene encodes an enzyme in the jasmonic acid synthesis pathway. Plant Cell 12 1041–1061 [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Schilmiller AL, Koo AJ, Howe GA (2007) Functional diversification of acyl-coenzyme a oxidases in jasmonic acid biosynthesis and action. Plant Physiol 143 812–824 [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Shan X, Wang Z, Xie D (2007) Jasmonate signal pathway in Arabidopsis. J Integr Plant Biol 49 81–86 [Google Scholar]
  36. Shan X, Zhang Y, Peng W, Wang Z, Xie D (2009) Molecular mechanism for jasmonate-induction of anthocyanin accumulation in Arabidopsis. J Exp Bot 60 3849–3860 [DOI] [PubMed] [Google Scholar]
  37. Staswick PE, Su WP, Howell SH (1992) Methyl jasmonate inhibition of root-growth and induction of a leaf protein are decreased in an Arabidopsis thaliana mutant. Proc Natl Acad Sci USA 89 6837–6840 [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Stintzi A, Browse J (2000) The Arabidopsis male-sterile mutant, opr3, lacks the 12-oxophytodienoic acid reductase required for jasmonate synthesis. Proc Natl Acad Sci USA 97 10625–10630 [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Sun J, Xu Y, Ye S, Jiang H, Chen Q, Liu F, Zhou W, Chen R, Li X, Tietz O, et al (2009) Arabidopsis ASA1 is important for jasmonate-mediated regulation of auxin biosynthesis and transport during lateral root formation. Plant Cell 21 1495–1511 [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Szekeres M, Nemeth K, Koncz-Kalman Z, Mathur J, Kauschmann A, Altmann T, Redei GP, Nagy F, Schell J, Koncz C (1996) Brassinosteroids rescue the deficiency of CYP90, a cytochrome P450, controlling cell elongation and deetiolation in Arabidopsis. Cell 85 171–182 [DOI] [PubMed] [Google Scholar]
  41. Tanaka K, Asami T, Yoshida S, Nakamura Y, Matsuo T, Okamoto S (2005) Brassinosteroid homeostasis in Arabidopsis is ensured by feedback expressions of multiple genes involved in its metabolism. Plant Physiol 138 1117–1125 [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Tang WQ, Kim TW, Oses-Prieto JA, Sun Y, Deng ZP, Zhu SW, Wang RJ, Burlingame AL, Wang ZY (2008) Brassinosteroid-signaling kinases(BSKs) mediate signal transduction from the receptor kinase BRI1 in Arabidopsis. Science 321 557–560 [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Thines B, Katsir L, Melotto M, Niu Y, Mandaokar A, Liu G, Nomura K, He SY, Howe GA, Browse J (2007) JAZ repressor proteins are targets of the SCF(COI1) complex during jasmonate signalling. Nature 448 661–665 [DOI] [PubMed] [Google Scholar]
  44. Tiryaki I, Staswick PE (2002) An Arabidopsis mutant defective in jasmonate response is allelic to the auxin-signaling mutant axr1. Plant Physiol 130 887–894 [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Wang Z, Dai L, Jiang Z, Peng W, Zhang L, Wang G, Xie D (2005) GmCOI1, a soybean F-box protein gene, shows ability to mediate jasmonate-regulated plant defense and fertility in Arabidopsis. Mol Plant Microbe Interact 18 1285–1295 [DOI] [PubMed] [Google Scholar]
  46. Wang ZY, Wang Q, Chong K, Wang F, Wang L, Bai M, Jia C (2006) The brassinosteroid signal transduction pathway. Cell Res 16 427–434 [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Wasternack C (2007) Jasmonates: an update on biosynthesis, signal transduction and action in plant stress response, growth and development. Ann Bot (Lond) 100 681–697 [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Xiao S, Dai L, Liu L, Wang Z, Peng W, Xie D (2004) COS1: an Arabidopsis coronatine insensitive 1 suppressor essential for regulation of jasmonate-mediated plant defense and senescence. Plant Cell 16 1132–1142 [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Xie DX, Feys BF, James S, Nieto-Rostro M, Turner JG (1998) COI1: an Arabidopsis gene required for jasmonate-regulated defense and fertility. Science 280 1091–1094 [DOI] [PubMed] [Google Scholar]
  50. Xu L, Liu F, Lechner E, Genschik P, Crosby WL, Ma H, Peng W, Huang D, Xie D (2002) The SCF(COI1) ubiquitin-ligase complexes are required for jasmonate response in Arabidopsis. Plant Cell 14 1919–1935 [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Yan J, Zhang C, Gu M, Bai Z, Zhang W, Qi T, Cheng Z, Peng W, Luo H, Nan F, et al (2009) The Arabidopsis CORONATINE INSENSITIVE1 protein is a jasmonate receptor. Plant Cell 21 2220–2236 [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Yan Y, Stolz S, Chetelat A, Reymong P, Pagni M, Dubugnon L, Farmer EE (2007) A downstream mediator in the growth repression limb of the jasmonate pathway. Plant Cell 19 2470–2483 [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Zimmermann P, Hirsch-Hoffmann M, Hennig L, Gruissem W (2004) GENEVESTIGATOR: Arabidopsis microarray database and analysis toolbox. Plant Physiol 136 2621–2632 [DOI] [PMC free article] [PubMed] [Google Scholar]

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