Tomato BRASSINOSTEROID INSENSITIVE1 Is Required for Systemin-Induced Root Elongation in Solanum Pimpinellifolium but Is Not Essential for Wound Signaling W

The Plant Cell, Vol. 19: 1709–1717, May 2007, www.plantcell.org ª 2007 American Society of Plant Biologists

Tomato BRASSINOSTEROID INSENSITIVE1 Is Required for Systemin-Induced Root Elongation in Solanum pimpinellifolium but Is Not Essential for Wound Signaling W

Nicholas Holton,a,b Ana Can˜ o-Delgado,c,1 Kate Harrison,a,b Teresa Montoya,b Joanne Chory,c and Gerard J. Bishopa,b,2 a Division of Biology, Imperial College London, Ashford, Kent TN25 5AN, United Kingdom b Institute of Biological Sciences, University of Wales, Aberystwyth SY23 3DA, United Kingdom c Howard Hughes Medical Institute and Plant Biology Laboratory, Salk Institute for Biological Studies, La Jolla, California 92037

The tomato Leu-rich repeat receptor kinase BRASSINOSTEROID INSENSITIVE1 (BRI1) has been implicated in both peptide (systemin) and steroid (brassinosteroid [BR]) hormone perception. In an attempt to dissect these signaling pathways, we show that transgenic expression of BRI1 can restore the dwarf phenotype of the tomato curl3 (cu3) mutation. Conﬁrmation that BRI1 is involved in BR signaling is highlighted by the lack of BR binding to microsomal fractions made from cu3 mutants and the restoration of BR responsiveness following transformation with BRI1. In addition, wound and systemin responses in the cu3 mutants are functional, as assayed by proteinase inhibitor gene induction and rapid alkalinization of culture medium. However, we observed BRI1-dependent root elongation in response to systemin in Solanum pimpinellifolium.In addition, ethylene perception is required for normal systemin responses in roots. These data taken together suggest that cu3 is not defective in systemin-induced wound signaling and that systemin perception can occur via a non-BRI1 mechanism.

INTRODUCTION tomato (Solanum lycopersicum), the curl3 (cu3) mutant has been characterized as a BR-insensitive dwarf (Koka et al., 2000), and a Brassinosteroids (BRs) are plant steroid hormones required for candidate gene approach showed that this mutant was defective normal growth and development. Mutants in BR biosynthesis or in a homolog of BRI1 (Montoya et al., 2002). signaling have a characteristic dramatic dwarf phenotype, indi- Tomato BRI1 also has been purified as a systemin binding cating the importance of these hormones in plant growth and protein (Scheer and Ryan, 2002). Systemin is an 18–amino acid development (for reviews on BRs and BR signaling, see Bishop, protein that is proteolytically cleaved from a 200–amino acid 2003; Vert et al., 2005; Szekeres and Bishop, 2006). The major precursor (Pearce et al., 1991; McGurl et al., 1992). This peptide BR receptor in Arabidopsis thaliana is encoded by the BRASSI- is able to induce the production of protease inhibitors (PINs), and NOSTEROID INSENSITIVE1 (BRI1) gene, which when mutated, plants that have reduced systemin levels are more susceptible to results in a dwarf plant, similar in stature to BR biosynthesis insect herbivory (Orozco-Ca´ rdenas et al., 1993). Interestingly, mutants (Clouse et al., 1996). BRI1 encodes a receptor Ser/Thr systemin appears to be a Solanaceae-specific protein and has kinase with a predicted extracellular domain of ;24 Leu-rich only been identified in certain Solanaceae tribes (e.g., solanum repeats (LRRs) that is interrupted by an island domain between and capsicum) (Constabel et al., 1998). The putative receptor repeat 20 and 21 (Li and Chory, 1997; Vert et al., 2005). This is for systemin was isolated biochemically using a radiolabeled followed by a transmembrane domain and a C-terminal kinase (Li photoaffinity systemin that bound to a 160-kD protein named and Chory, 1997; Vert et al., 2005). BRI1 binds BRs via a novel systemin receptor 160 (SR160). Cloning of SR160 revealed that it steroid binding domain of ;100 amino acids that encompasses was a BRI1 homolog (Scheer and Ryan, 2002). To confirm that Sl LRR21 and the island domain (Kinoshita et al., 2005; Vert et al., BRI1 (SR160) was the systemin receptor, cu3 mutants were 2005). BRI1 orthologs are known in several plant species. In assayed for their response to systemin. Although cu3 mutants are predicted to have a null allele (nonsense mutation in LRR24; 1 Current address: Gene´ tica Molecular, CONSORCIO Consejo Superior Montoya et al., 2002), they exhibit only a 50% reduction in their de Investigaciones Cientificas–Institut de Recerca i Tecnologia Agro- response to systemin via measurement of PIN gene induction alimenta` ries, C/Jordi Girona 18-26, Barcelona 08034, Spain. 2 To whom correspondence should be addressed. E-mail g.bishop@ (Scheer et al., 2003). Tobacco (Nicotiana tabacum), another imperial.ac.uk; fax 44-207-584-2056. solanaceous species, lacks a closely related systemin sequence, The author responsible for distribution of materials integral to the and transformation of SR160 into tobacco produced the pre- findings presented in this article in accordance with the policy described dicted systemin-specific binding and alkalinization response in the Instructions for Authors (www.plantcell.org) is: Gerard J. Bishop (Scheer et al., 2003). Taken together, these data suggested ([email protected]). W Online version contains Web-only data. that Sl BRI1 (SR160) was a dual ligand receptor for systemin and www.plantcell.org/cgi/doi/10.1105/tpc.106.047795 brassinolide (BL). 1710 The Plant Cell

Here, we provide further proof that Sl BRI1 is a functional BR receptor. In addition, we demonstrate that both cu3 and the BR synthesis mutant extreme dwarf (dx) are capable of producing systemin and wound responses. Intriguingly, we found that Solanum pimpinellifolium roots elongate in response to systemin, in a BRI1-dependent fashion. However, systemin-induced root elongation was not observed in closely related species. In these species, root length was reduced, which in S. lycopersicum is both jasmonate and BR independent but is ethylene dependent. These results indicate that Sl BRI1 is not essential for systemin-induced wound response, although in certain species, it may promote root growth.

RESULTS

Genetic Complementation of the cu3 Phenotype

Previous analysis of the cu3 dwarf mutant demonstrated that it is insensitive to BL and that Spi BRI1 (S. pimpinellifolium BRI1)is mutated (Montoya et al., 2002). To confirm that Spi BRI1 is responsible for the cu3 phenotype, complementation was performed by introduction of Sl BRI1 (L. esculentum BRI1) under the control of the cauliflower mosaic virus (CaMV) 35S promoter (35S:Sl BRI1). Introduction of this T-DNA construct into the cu3 background generated two lines that recovered the dwarf phenotype (Figure 1). The 35S:Sl BRI1 plants exhibited additional phenotypes, including delayed germination by 1 to 2 d, compared with the wild type or cu3. Internodal distance and hypocotyl length were greater than wild-type plants, and lateral branching (side shoot length) was also reduced (see Supple- mental Table 1 online). Although Arabidopsis plants overex- pressing At BRI1 have elongated petioles, this was not observed in 35S:Sl BRI1 plants. The leaves were more ovoid, with reduced serration, than those of the wild type. Leaves also frequently developed necrotic patches, reminiscent of a hypersensitive response. However, no pathogen could be associated with this phenotype, and wild-type plants did not exhibit a similar pheno- Figure 1. Sl BRI1 Complements the the cu3 Phenotype. type, even following inoculation with 35S:Sl BRI1 leaf extracts. cu3 plants were transformed with Sl BRI1 under the control of the CaMV Complementation of cu3 with Sl BRI1 under the control of its 35S promoter or the Sl BRI1 promoter. native promoter restored plants to the wild type and did not (A) Three-week-old seedlings of the wild type, cu3, and 35S:Sl BRI1 in exhibit the additional phenotypes observed with 35S:Sl BRI1 the cu3 background. (Figure 1). (B) Three-week-old seedlings of the wild type, cu3, and pSl BRI1:Sl BRI1 Morphological analyses showed that the introduction of 35S:Sl (native promoter) in the cu3 background. (C) and (D) Leaves were photographed from 6-week-old plants of cu3 BRI1 restored the BL responses abolished by the cu3 mutation. ([C]; inset, wild type) and 35S:Sl BRI1 (D). To confirm this, seedlings were treated with BL and root length was recorded (Figure 2). As had been previously demonstrated, cu3 roots were insensitive to BL treatment (Koka et al., 2000; promoter (Table 1; see Supplemental Figure 1 online). Partially Montoya et al., 2002). The introduction of 35S:Sl BRI1 restored a complementing lines had longer hypocotyls, increased rosette BL-responsive reduction in root elongation, with the sensitivity to area, and longer inflorescence stems compared with bri1-5, BL being similar to wild-type plants. although they were smaller than wild-type plants. To determine if the failure of Sl BRI1 to complement the Arabidopsis mutant was Sl BRI1 Does Not Fully Complement Arabidopsis bri1-5 specific to Sl BRI1 or common to other solanaceous species, the tobacco and potato homologs Nb BRI1 (Nicotiana benthamiana The results shown here and previously strongly indicated that Sl BRI1) and St BRI1 (Solanum tuberosum BRI1), respectively, were BRI1 is the functional ortholog of At BRI1. However, when Sl transformed into bri1-5. Both of these genes failed to fully BRI1 was transformed into the At BRI1 mutant bri1-5, it failed to complement the bri1-5 mutant phenotype, although both mor- fully complement this mutation when placed under the control of phologically recovered the cu3 phenotype. Interestingly, At BRI1 either the Arabidopsis BRI1, S. lycopersicum BRI1, or CaMV 35S morphologically restores the cu3 phenotype to almost the wild Tomato BRI1 and Systemin Signaling 1711

(Figure 4B). To conﬁrm this result, systemin treatments were performed with another tomato BRI1 mutant, abs, and systemin- induced PINII transcript accumulation was also found to be similar to wild-type plants (Figure 4B). To test whether BRs are required for PINII transcript accumulation, the BR synthesis mutant dx, which lacks bioactive BRs in vegetative tissue (Bishop et al., 1999), was examined for its response to systemin. The dx mutants had wild-type levels of PINII expression, indicating that bioactive BRs are not required for systemin-mediated PIN gene induction.

Figure 2. Sl BRI1 Restores BL Signaling. cu3 Is Not Compromised in Wound-Induced PINII Expression The root length of wild-type, cu3, and 35S:Sl BRI1 seedlings was measured in response to BL at 7 d after germination. Error bars indicate Systemin induction is associated with mechanical wounding, SE of the mean (n ¼ 10). and it may be expected that mutants in the systemin receptor would be defective in their wound response. cu3 mutants were type, indicating that a more stringent sequence requirement in mechanically wounded, and this treatment induced systemic Arabidopsis is required for genetic complementation. PINII expression (Figure 5). In addition, systemic accumulation of To further investigate why Sl BRI1 failed to rescue bri1-5, PINII transcripts was found in the Sl BRI1 mutant abs and in the x chimeric constructs between At BRI1 and Sl BRI1 were pro- BR synthesis mutant d . PINII transcripts were detectable be- duced. Complementation was achieved when the kinase domain tween 2 and 4 h after wounding, and no signiﬁcant difference was of Sl BRI1 (amino acids 749 to 1207) was fused to the extracel- observed in the timing of the induction or the duration of re- lular LRR region of At BRI1 (amino acids 1 to 739). However, the sponse in any of the mutants. The level of PINII accumulation in reciprocal construct failed to restore the bri1-5 mutant pheno- all mutants was greater than the wild type, which may be a type (Table 1). consequence of leaf morphology rather than alteration in wound responsiveness.

BL Binding Is Not Detectable in cu3 Microsomes

Having established that expression of Sl BRI1 restores stature MediumAlkalinizationinResponsetoSysteminIsNot and BL responsiveness to cu3, we asked whether cu3 mem- Altered in cu3 3 brane fractions contained BL binding activity (Figure 3). In [ H]- The alkalinization of cell suspension medium has been used to BL binding assays using total microsomal membranes, a greatly show the activity of systemin (Meindl et al., 1998). This response reduced BL binding activity was observed in cu3 mutants. The is not affected by BL, although overexpression of tomato BRI1 in dissociation constant (Kd) observed in wild-type microsomal tobacco suspension enables systemin-responsive alkalinization membranes was ;28 nM, which is similar to values obtained in (Scheer et al., 2003). Suspension cultures of the wild type and cu3 Arabidopsis (Wang et al., 2001). In agreement with the genetic were generated, and the pH of the medium was recorded after data, these results demonstrated in vivo that wild-type tomato Sl the addition of systemin (Figure 6). Both mutant and wild-type BRI1 receptor protein is necessary for BL binding.

Table 1. Complementation of bri1-5 with BRI1 Systemin-Induced PINII Expression Does Not Require a b b BRI1 or BR At BRI1 Promoter Wild Type Dwarf At BRI1 11 23 PIN gene induction and rapid alkalization of cell suspension Sl BRI1 0 118 culture medium are key assays in determining the bioactivity of St BRI1 0 41 systemin (Meindl et al., 1998). Scheer et al. (2003) showed that Nb BRI1 0 37 cu3 had a reduced ability to induce the accumulation of PIN At LRR-LeKD 6 11 protein following treatment with either methyl jasmonate or Sl LRR-AtKD 0 17 systemin. Systemin treatments of 35S:Sl BRI1, cu3, and wild- Sl BRI1 promoter type seedlings were performed to conﬁrm the BRI1 dependence At BRI1 54 72 of systemin-induced PINII expression. During these studies, Sl BRI1 0 312 we noted that the rate of systemin solution uptake in cu3 was 35S promoter signiﬁcantly lower than the wild type (Figure 4A). This was pre- Sl BRI1 0 8 Empty vector 0 48 sumably due to a lower transpiration rate in cu3 mutants because of the reduced leaf area. Systemin treatments were performed so a BRI1 genes were under the control of Sl BRI1 or At BRI1 promoters. b that all seedlings were treated with the same amount of systemin, Number of bri1-5 transformants with either wild-type or dwarf (bri1-5) and no difference in the level of PINII transcripts or the sensitivity morphology. Only plants that were fully complemented were scored as wild type. to systemin was observed between the wild type and mutants 1712 The Plant Cell

dependent and required >1 nM systemin to elongate roots in the presence of 1 nM BL. This indicated that BRI1 plays a role in root elongation and that its function impacts both the systemin and BL signaling pathways. The interaction between systemin and BL responses was examined further by investigating the effect of BL on the root growth of seedlings expressing reduced levels of prosystemin. Roots of antisense prosystemin plants responded in a similar way as the wild type (see Supplemental Figure 3 online). This was not unexpected since systemin is not expressed in roots (McGurl et al., 1992; N. Holton, unpublished data). The antisense line used was in an S. lycopersicum cultivar, and Figure 3. BL Binding Is Diminished in cu3 Microsomal Fractions. this species exhibited reduced root length in response to systemin (P < 0.001 for systemin concentrations >0.1 nM) (Figure Mature tomato wild-type and cu3 leaves of mature plants were used to 7). This result was opposite to that observed in S. pimpinellifolium assess [3H]-BL binding in vivo. Microsomal extracts from fresh tissues roots. However, similar to S. pimpinellifolium roots, those of were incubated in 2, 10, 25, 50, 75, or 100 nM [3H]-BL. Speciﬁc [3H]-BL binding is reduced in the cu3 microsomal extracts compared with the S. lycopersicum had decreased root hair growth and lateral wild type. Data represent the average measurements of three indepen- branching in the presence of systemin. dent experiments. Error bars indicate SE of the mean. cultures responded at similar times and with similar amplitudes (P > 0.1).

Tomato Roots Respond to Systemin

To discern the possible involvement of systemin in the BR response, we used root elongation to monitor the bioactivity of various systemin and hormone treatments. Root elongation was used because it is known to be a sensitive method to measure BR bioactivity in tomato (Roddick, 1994) and because cu3 mutants are known to be defective in the inhibition of root elongation in response to BRs (Koka et al., 2000; Montoya et al., 2002). Wild- type and cu3 seedlings were grown on medium containing systemin, and root length was recorded (Figure 7). When wild- type S. pimpinellifolium seedlings were grown on systemin, roots were longer than those of untreated seedlings (P < 0.001 for all systemin concentrations). In addition, lateral branching and root hair growth were inhibited. Interestingly, cu3 seedlings exhibited a dose-dependent inhibition of root elongation (P < 0.001 for systemin concentrations >0.1 nM). Root hair growth in these mutants was not suppressed by the addition of systemin. In addition, an anthocyanin-like coloration was observed in cu3 roots at systemin concentrations >10 nM. The involvement of BRI1 in this response was conﬁrmed by systemin treatment of cu3 plants transformed with Sl BRI1 (Figure 8). These plants showed a restoration of wild-type systemin response. As a Figure 4. BR Mutants Can Induce PINII in Response to Systemin. control for these experiments, the biologically inactive Ala-17 (A) Reduced transpiration rate in cu3. Seedlings were cut through the systemin was used and did not exhibit any signiﬁcant bioactivity stem and placed in 250 mL of 10 nM systemin and allowed to take up the in roots (P > 0.05; see Supplemental Figure 2 online). solution. Each hour seedlings were removed and the volume of solution The inhibition of root growth in cu3 indicates that BRI1 is recorded. Six seedlings were used for each time point. Evaporation of required for a wild-type response to systemin in this assay. To solution was also recorded (empty). Error bars indicate SE of the mean. (B) RNA gel blot analysis of PINII expression in response to systemin further investigate the role of BR in this systemin response, applied at 0, 0.1, 1, 10, and 100 nM. Leaf samples were taken 8 h after treatments with systemin in the presence of BL were performed systemin treatment. Membranes were probed with PINII (top) and (Figure 8). Not surprisingly, the response to systemin in the reprobed with an 18S rRNA probe (bottom) as a loading control. The BR-insensitive mutant cu3 was not altered by the addition of BL. top PINII blot was exposed for 4 h and the bottom blot for 16 h. S. In wild-type seedlings, systemin partially suppressed the pimpinellifolium (wt, top panel), cu3, 35S:Sl BRI1, S. lycopersicum (wt, BL-induced reduction in root length. This response was dose bottom panel), dx,andabs. Tomato BRI1 and Systemin Signaling 1713

DISCUSSION

The role of BRI1 in BR perception in Arabidopsis is well established. As such, the purification of tomato BRI1 by binding to systemin has been an enigma (Yin et al., 2002; Bishop, 2003; Szekeres, 2003; Boller, 2005). It was therefore important to show that Sl BRI1 was a functional BR receptor using both genetic and biochemical approaches. Genetic restoration of the cu3 mutant phenotype using the CaMV 35S promoter to express the tomato BRI1 generated nondwarf plants that had elongated stems, indicating a role for tomato BRI1 in growth. The rescued lines also exhibited less serration of the leaf margins, indicating a role for BRs in leaf margin development. This is not surprising since Figure 5. Wound-Induced PINII Expression Is Not Compromised in BR Arabidopsis mutants in BR biosynthesis and signaling have been Mutants. identified in screens for leaf phenotypes (Kim et al., 1998; Pe´ rez- Pe´ rez et al., 2002). Transformation experiments using the en- Wounding was performed on S. pimpinellifolium, cu3, 35S:Sl BRI1, S. dogenous Sl BRI1 promoter to express the Sl BRI1 gene resulted lycopersicum, dx, and abs plants (see Methods). RNA was extracted from in plants that have phenotypes very similar to those of the wild systemic unwounded leaves at 0, 2, 4, 8, and 24 h after wounding. Membranes were probed with PINII (top) and reprobed with an 18S rRNA type, providing further proof that the cu3 mutant is defective in probe (bottom) as a loading control. the LRR receptor kinase Sl BRI1. Sl BRI1, Nb BRI1, and St BRI1 are able to phenotypically rescue the cu3 phenotype to the wild type. However, these genes Since BR mutants exhibit defective etiolation, we analyzed the fail to fully complement the Arabidopsis BRI1 mutant bri1-5 root response to systemin in both light- and dark-grown seed- when expressed under the control of the Arabidopsis BRI1 lings. When systemin treatments of S. pimpinellifolium and cu3 promoter. When the extracellular domain of the Arabidopsis were performed on etiolated seedlings, root length was de- gene is fused to the tomato kinase domain, complementation creased in the presence of systemin (Figure 7B). We also noticed is achieved; this suggests that the extracellular LRR region that the root length of untreated etiolated S. pimpinellifolium from Arabidopsis is required for full function in Arabidopsis. seedlings was significantly longer than light-grown seedlings; The extracellular region of BRI1 is poorly conserved between however, this was not the case for cu3 (P < 0.001 and P > 0.1, species when compared with the kinase domain. However, respectively). These data indicated that the response to systemin several amino acids are conserved in this region in all known was different between S. lycopersicum and S. pimpinellifolium BRI1 homologs. Of particular interest is the NGSM motif located species (i.e., S. lycopersicum seedlings lacked root elongation). in the region that has recently been shown to bind BRs (Kinoshita However, when grown in the dark, S. pimpinellifolium seedlings et al., 2005; see Supplemental Figure 4 online). This motif is found responded in a similar manner to light-grown S. lycopersicum in all known BR binding proteins, BRI1 and related BRL1 and seedlings, in that both sets of seedlings have reduced root length BRL3 (Can˜ o-Delgado et al., 2004), but not in the closely related in the presence of systemin. At BRL2 and Os BRL2. At BRL2 and Os BRL2 do not bind BR or Further investigation of the role of BRI1 in the response to restore BR responsiveness (Nakamura et al., 2006). This motif is systemin was performed using the weak BRI1 mutant abs (S. lycopersicum). In this mutant, systemin induced a response similar to wild-type roots. In addition, the BL synthesis mutant dx produced a response equivalent to the wild type. These data suggested that the systemin-induced root inhibition of S. lycopersicum seedlings is not influenced by BR signaling, but it is in S. pimpinellifolium seedlings. Overexpression of prosystemin results in jasmonate-dependent increased hypocotyl elongation (Howe and Ryan, 1999). To determine if the systemin-induced changes in root length are also jasmonate dependent, the responses of jasmonate synthesis mutants def1 and spr2 (Li et al., 2003) (S. lycopersicum mutants) were investigated. Both mutants had a wild-type reduction in root length when grown on systemin (Figure 8), which indicated that the reduced root growth was independent of jasmonate Figure 6. Medium Alkalinization Induced by Systemin. signaling. Ethylene is also known to be involved in systemin Suspension cells of the wild type and cu3 were treated with 100 pM responses. Root lengths in the ethylene-insensitive mutant Never systemin and the pH of the suspension recorded. Mock treatment of the ripe (Nr) were not significantly different in response to systemin wild type had water added instead of systemin. Data represent the (P > 0.05). This indicates that in S. lycopersicum, the reduction in average measurements of three samples from one representative ex- root length is dependent on ethylene signaling. periment. Error bars indicate SE of the mean. 1714 The Plant Cell

found in Sl BRI1; therefore, it is not surprising that the cu3 mutants defective in Sl BRI1 had reduced BL binding. To study the putative interaction of BR and systemin signaling, we sought conﬁrmation that cu3 mutants were defective in systemin signaling. In previous studies, the cu3 mutant was shown to have only ;50% of wild-type PIN gene induction (Scheer et al., 2003), even though this is a null mutant. To help clarify, we assayed PINII mRNA accumulation using a range of systemin concentrations and altering the length of time for systemin uptake in the mutant lines to allow equal systemin loading. Following such systemin treatments, bri1 mutants showed PINII induction equivalent to the wild type, indicating that the previously reported reduced level of PIN accumulation may have been the consequence of the absolute amount of systemin that the mutant was exposed to. It could be argued that due to the longer exposure of the mutants to systemin that the relative concentration in the cu3 mutants may be higher than that in wild-type plants. Without measuring systemin levels in the plants this will be difﬁcult to resolve. However, this does not detract from the fact that the tomato bri1 mutants are able to respond to systemin and that this suggests the presence of an additional mechanism for systemin perception. These results are consistent with the observation that dx, cu3, and abs mutants exhibit a wound response. This response is greater than in wild-type plants, and this is most likely the consequence of altered plant morphology. However, the possi- bility exists that BRI1 and BRs are involved in the regulation

Figure 8. Root Elongation in Response to Systemin and BL.

Seedlings were grown with or without treatment and root length was recorded after 7 d. (A) Seedlings grown with varying concentrations of BL with or without 10 nM systemin treatment. (B) Seedlings were grown on medium containing different levels of systemin with or without 1 nM BL. (C) Response of mutant and Sl BRI1 transformed plants to systemin. Seedlings were grown on medium with or without the addition of 10 nM Figure 7. Root Elongation in Response to Systemin. systemin and root length recorded. Plants analyzed are S. pimpinellifolium, cu3, pSl BRI1:Sl BRI1 transformed into cu3, S. lycopersicum cv (A) Seedlings were grown on medium containing 0, 0.1, 1, 10, or 100 nM Moneymaker (MM), S. lycopersicum cv Ailsa Craig (AC), S. lycopersicum systemin for 7 d (S. pimpinellifolium and cu3)or5d(S. lycopersicum cv cv Castlemart (CM), dx, abs, def1, spr2,andNr. Asterisks indicate Moneymaker). Root length was then measured. Data are expressed as P < 0.001. Error bars indicate SE from the mean (n 10). percentage change of untreated seedlings. ¼ (B) S. pimpinellifolium and cu3 seedlings were light or dark grown on medium with or without 10 nM systemin for 7 d. Error bars indicate SE of the mean (n ¼ 10). Tomato BRI1 and Systemin Signaling 1715

of wound-induced PINII induction. Taken together, these data 2006), the fact that both systemin and At PEP1 bind to an LRR suggested that a functional BRI1 receptor is not essential for the Se/Thr kinase suggests that their mode of action is similar. These systemin-induced wound response. Furthermore, systemin treat- findings indicate that further investigation into systemin percep- ments of suspension cultures from cu3 mutants and the wild type tion is required to clarify whether conserved mechanisms of confirmed that medium alkalinization in response to systemin peptide hormone signaling exist in plants and may also define was normal. Thus, Sl BRI1 is not essential for the systemin- systemin’s role in plant development. induced alkalinization response. However, Scheer et al. (2003) demonstrated that the introduction of Spv BRI1 into tobacco suspension cultures resulted in alkalinization in response to METHODS systemin. This response does not occur in untransformed cultures and therefore indicates that in tobacco, Spv BRI1 facilitates T-DNA Construction and Generation of Complemented Lines alkalinization in response to systemin. In S. pimpinellifolium, Sl BRI1 was PCR amplified from Solanum lycopersicum cv Moneymaker however, an additional systemin receptor is present that enables using primers 59-CATCAAGAGCTCAAGCTATAGATTCAAG-39 to introduce the alkalinization response in cu3 mutants. an SstI site in the 59 untranslated leader of Sl BRI1 and 59-TGGATGG- BRs cause cell elongation, and although the overexpression of GAACTAGTGGTACATAC-39 that introduced an SpeI site in the 39 un- prosystemin results in plants with jasmonate-dependent elon- translated tail. The PCR product was cloned and sequenced. The gated hypocotyls (Howe and Ryan, 1999), further clarification of resulting plasmid was digested with SstIandSpeI, and Sl BRI1 was systemin-induced elongation was sought. Systemin was found cloned into T-DNA vector GB1421 (Bishop et al., 1999) cut with SstIand to increase S. pimpinellifolium root length, but in cu3 mutants, it XbaI to generate 35S:Sl BRI1. inhibited root elongation, indicating the presence of both BRI1- To produce BRI1 constructs under the control of the S. lycopersicum dependent and -independent systemin signaling. In addition, promoter, a 2-kb promoter region was amplified from the tomato BAC treatment of wild-type roots with both systemin and BL suggests LeHBa28J23 (Clemson University Genomics Institute) using the primers 59-GAATTCTAGAGAGGGAAACATCGT-39 and 59-GGTACCGAAACTTT- some antagonism. ATAGCTTAAATGGTG-39. The promoter region was then cloned into the When etiolated S. pimpinellifolium or light-grown S. lycopersi- EcoRI/KpnI sites of pCA2302 (pCAMBIA2300 containing the EcoRI/BstUI cum roots were investigated, root length was reduced in re- green fluorescent protein fragment from pCAMBIA1302 cloned into the sponse to systemin, suggesting that both environmental and EcoRI/PmeI sites). A 1.8-kb Arabidopsis thaliana promoter was amplified species-specific factors influence the way in which roots re- using PCR primers (59-CAATTGGAGCGCGTGTAGACCACG-39 and 59-GGT- spond to systemin. The reduction in root length observed in ACCTTGTGAGAGAGAAAAGTGTG-39) and cloned as an MfeI/KpnI frag- S. lycopersicum was not altered in jasmonate synthesis mutants, ment into EcoRI/KpnI of pCA2302. BRI1 gene regions were then cloned unlike the response observed in hypocotyls of plants overex- into either vector as KpnI/SpeI fragments following amplification with the pressing prosystemin (Howe and Ryan, 1999). However, Nr root following PCR primers: Sl BRI1,59-GGTACCTTTGAAGATGAAAGC-39 length was not significantly altered by the addition of systemin, and 59-ACTAGTACCTCCAAGGTGTTTGCTCAG-39;AtBRI1,59-GGTAC- CTTGAGAAATGAAGAC-39 and 59-ACTAGTTCCACCTAATTTTCCTTC- suggesting that ethylene perception is required for the reduction AGG-39.TheSlBRI1/At BRI1 fusion constructs were produced by in root length observed. Taken together, these results imply that amplifying both the LRR and kinase regions and introducing an internal an interaction between ethylene signaling and signaling via BRI1 Kpn2I restriction site. This was performed using terminal primers de- plays a key role in the systemin-induced root growth observed. scribed above and the following internal primers: Sl BRI1,59-TCA- Environmental (e.g., light) and species-specific factors influence TTCCGGAGAGATTATTGTTTGACAGGTCAATC-39 and 59-AATCTCTC- the interaction between the two pathways determining whether CGGAATGATTCCTGAATCTGCACC-39;AtBRI1,59-TTGTCCGGACCG- there is inhibition or stimulation of root elongation. ATTCCTGAGATGGGTC-39 and 59-TCGGTCCGGACAAATTATTATTCG-39. The interaction between ethylene and BR signaling is not well These fragments were then sequentially ligated into the binary vectors understood; however, an interaction has been observed. BRs containing either promoter. The Nicotiana benthamiana and Solanum can stimulate ethylene biosynthesis, and ethylene induces ex- tuberosum cv Maris Peer BRI1 genes were amplified by PCR (59-TCG- GTACCTTTGAAGATGAAACCTCACAAGAG-39 and 59-TCACTAGTACCT- pression of BR synthesis genes (Schlagnhaufer and Arteca, 1985; CCTAGGTGTTTGCTCAGCTCATTA-39;59-GGTACCTTTGAAGATGAAAGC-39 De Grauwe et al., 2005). BR synthesis mutants fail to develop the and 59-ACTAGTACCTCCAAGGTGTTTGCTTAGCTCATTGCC-39,respec- characteristic apical hook in response to the ethylene precursor tively) and cloned as KpnI/SpeI fragments under the control of the 1-aminocyclopropane-1-carboxylic acid (De Grauwe et al., Arabidopsis promoter. All constructs were sequenced to ensure no 2005); however, BRI1 and BR synthesis mutants are not altered mutations were introduced during PCR. in ethylene sensitivity (Clouse et al., 1996; Mussig et al., 2003). All constructs were transformed into cu3 as described previously Given the complexity of BR/ethylene interactions, further inves- (Bishop et al., 1999; Montoya et al., 2005). Constructs were transformed tigation of the influence of systemin in these pathways is required. into Arabidopsis bri1-5 by floral dipping as described by Clough and Bent Systemin is a member of the growing number of peptides that (1998). have been shown to have biological activity in plants that include tobacco systemins, CLAVATA3, S-locus protein 11, phytosulfo- 3H-BL Binding to Microsomal Fractions kine, and RALFs (Matsubayashi and Sakagami, 2006). One newly The microsomal fractions for 3H-BL experiments were extracted from identified Arabidopsis peptide, At Pep1 (Huffaker et al., 2006), fresh leaves of wild-type and cu3 mature plants as described by Kinoshita has been shown to elicit plant defense responses. Interestingly, et al. (2005). Protein concentration was determined (Protein Assay; Bio- At PEP1 binds an LRR kinase, PEPR1. While systemin does not Rad) and samples resuspended to 1 mg/mL in MES binding buffer (10 mM compete with binding of At PEP1 to PEPR1 (Yamaguchi et al., Mes-KOH, pH 5.7, 5 mM MgCl2, 0.1 mM CaCl2, and 0.25 M mannitol). 1716 The Plant Cell

Each BL binding assay contained 200 mL membrane suspensions in 2, 10, Supplemental Figure 1. Phenotypes of bri1-5 Transgenic Plants. 25, 50, 75, or 100 nM [3H]-BL, without or with 100-fold excess unlabeled Supplemental Figure 2. Ala-17–Substituted Systemin Is Biologically BL or indicated amount of unlabeled steroids, 1 mg mL1 BSA, and BL Inactive. binding buffer in 1000 mL total volume. The binding reactions were incubated for 30 min at 258C. The bound and free [3H]-BL were separated Supplemental Figure 3. BL Response of Antisense Prosystemin by filtering the mixture through a glass-fiber filter (Whatman; GF/F) and Roots. washing three times with 50 mL ice-cold BL binding buffer. Bound BL was Supplemental Figure 4. Sequence Alignment of the BL Binding quantified by scintillation counting as described by Wang et al. (2001). Region of BRI1 and Related Proteins. Supplemental Table 1. Characterization of cu3 Transformants. Systemin Application and Wounding

Systemin treatments were performed by cutting the stem of 3- to 4-week- old greenhouse-grown seedlings with a single-edge razor blade and then ACKNOWLEDGMENTS placing them in a systemin solution (peptides synthesized by Aﬁniti Research Products) or water for 1 h for the wild type (Solanum pimpi- We thank the glasshouse staff at both the Institute of Biological Sci- nellifolium or S. lycopersicum), abs,anddx or 3 h for cu3. After systemin ences, Aberystwyth, and the Division of Biology, Wye, for plant care. treatment, seedlings were transferred to water and samples taken 8 h This work has been generously supported by the Biotechnology and later. At least six seedlings were sampled for each treatment, and only the Biosciences Research Council (Grant P19961 to G.J.B.), by the Human two uppermost leaves were sampled. Treatments were performed in Frontier Research Program (G.J.B. and J.C.), and by the USDA (J.C.). continuous light. A.C.-D. was a long-term fellow of the Human Frontier Research Program. Mechanical wounding was performed on 4- to 5-week-old seedlings by We also thank C. Ryan and G. Howe for seeds of the tomato antisense crushing terminal leaﬂets with forceps three times. Samples were taken systemin line and jasmonate mutants, respectively, and J. Giovannoni from the leaf immediately above that which had been wounded. At least for the tomato BAC clone. six seedlings were used for each sample. RNA was extracted from systemin- and wound-treated samples, and RNA gel blotting was performed using either PINII or tomato 18S rRNA derived probes. Wounding Received September 29, 2006; revised March 29, 2007; accepted April and systemin treatments were performed at least three times with similar 27, 2007; published May 18, 2007. results.

REFERENCES Alkalinization Experiments

To analyze the alkalinization response of suspension cultures, S. pim- Bishop, G.J. (2003). Brassinosteroid mutants of crops. J. Plant Growth pinellifolium (wild type) and cu3 suspension cultures were produced from Regul. 22: 325–335. leaf-derived callus. Callus was maintained on Murashige and Skoog Bishop, G.J., Nomura, T., Yokota, T., Harrison, K., Noguchi, T., medium (Duchefa) containing 3% (w/v) sucrose, 3 mg/L 1-naphthalene Fujioka, S., Takatsuto, S., Jones, J.D.G., and Kamiya, Y. (1999). acetic acid, and 1.5 mg/L 6-benzylaminopurine. Callus was transferred to The tomato DWARF enzyme catalyses C-6 oxidation in brassinoste- liquid culture, in the same medium used for callus maintenance, and roid biosynthesis. Proc. Natl. Acad. Sci. USA 96: 1761–1766. subcultured every 7 d. Experiments were performed using 5 mL of culture Boller, T. (2005). Peptide signalling in plant development and self/non- 5 to 7 d after subculture. The pH of the culture medium was recorded prior self perception. Curr. Opin. Cell Biol. 17: 116–122. to and following the addition of systemin or water, in a volume of <10 mL. Can˜ o-Delgado, A., Yin, Y.H., Yu, C., Vafeados, D., Mora-Garcia, S., Experiments were repeated a total of three times. Two independent Cheng, J.C., Nam, K.H., Li, J.M., and Chory, J. (2004). BRL1 and culture lines of the wild type and cu3 were used. BRL3 are novel brassinosteroid receptors that function in vascular differentiation in Arabidopsis. Development 131: 5341–5351. Clough, S.J., and Bent, A.F. (1998). Floral dip: A simpliﬁed method for Root Elongation Assay Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant Seeds were surface sterilized with 10% commercial bleach, rinsed in J. 16: 735–743. water, and plated on one half-strength Murashige and Skoog medium Clouse, S.D., Langford, M., and McMorris, T.C. (1996). A brassinos- containing 1% (w/v) sucrose. At radical emergence, seeds were trans- teroid-insensitive mutant in Arabidopsis thaliana exhibits multiple ferred to 120-mm square Petri dishes containing the same medium as defects in growth and development. Plant Physiol. 111: 671–678. above with the addition of systemin or hormones. The plates were then Constabel, C.P., Yip, L., and Ryan, C.A. (1998). Prosystemin from potato, placed vertically and seedlings grown for 5 d for S. lycopersicum lines or black nightshade, and bell pepper: Primary structure and biological 7 d for S. pimpinellifolium lines. Seedlings were grown in a controlled activity of predicted systemin polypeptides. Plant Mol. Biol. 36: 55–62. environment at 248C with a 16-h daylength. Root length was then re- De Grauwe, L., Vandenbussche, F., Tietz, O., Palme, K., and Van Der corded for at least 10 seedlings per treatment. Experiments were re- Straeten, D. (2005). Auxin, ethylene and brassinosteroids: Tripartite peated at least twice, with similar results. control of growth in the Arabidopsis hypocotyl. Plant Cell Physiol. 46: 827–836. Howe, G.A., and Ryan, C.A. (1999). Suppressors of systemin signaling Accession Numbers identify genes in the tomato wound response pathway. Genetics 153: Sequence data from this article can be found in the GenBank/EMBL data 1411–1421. libraries under accession numbers EF471736 to EF471738. Huffaker, A., Pearce, G., and Ryan, C.A. (2006). An endogenous peptide signal in Arabidopsis activates components of the innate immune response. Proc. Natl. Acad. Sci. USA 103: 10098–10103. Supplemental Data Kim, G.-T., Tsukaya, H., and Uchimiya, H. (1998). The ROTUNDIFO- The following materials are available in the online version of this article. LIA3 gene of Arabidopsis thaliana encodes a new member of the Tomato BRI1 and Systemin Signaling 1717

cytochrome P-450 family that is required for the regulated polar Nakamura, A., et al. (2006). The role of OsBRI1 and its homologous elongation of leaf cells. Genes Dev. 12: 2381–2391. genes, OsBRL1 and OsBRL3, in rice. Plant Physiol. 140: 580–590. Kinoshita, T., Can˜ o-Delgado, A.I., Seto, H., Hiranuma, S., Fujioka, S., Orozco-Ca´ rdenas, M., Mcgurl, B., and Ryan, C.A. (1993). Expression Yoshida, S., and Chory, J. (2005). Binding of brassinosteroids to the of an antisense prosystemin gene in tomato plants reduces resistance extracellular domain of plant receptor kinase BRI1. Nature 433: 167–171. toward Manduca sexta larvae. Proc. Natl. Acad. Sci. USA 90: 8273– Koka, C.V., Cerny, R.E., Gardner, R.G., Noguchi, T., Fujioka, S., 8276. Takatsuto, S., Yoshida, S., and Clouse, S.D. (2000). A putative role Pearce, G., Strydom, D., Johnson, S., and Ryan, C.A. (1991). A for the tomato genes DUMPY and CURL-3 in brassinosteroid bio- polypeptide from tomato leaves induces wound-inducible proteinase synthesis and response. Plant Physiol. 122: 85–98. inhibitor proteins. Science 253: 895–898. Li,C.,Liu,G.,Xu,C.,Lee,G.I.,Bauer,P.,Ling,H.Q.,Ganal,M.W., Pe´ rez-Pe´ rez, J.M., Ponce, M.R., and Micol, J.L. (2002). The UCU1 and Howe, G.A. (2003). The tomato suppressor of prosystemin- Arabidopsis gene encodes a SHAGGY/GSK3-like kinase required for mediated responses2 gene encodes a fatty acid desaturase re- cell expansion along the proximodistal axis. Dev. Biol. 242: 161–173. quired for the biosynthesis of jasmonic acid and the production of a Roddick, J.G. (1994). Comparative root-growth inhibitory activity of 4 systemic wound signal for defense gene expression. Plant Cell 15: brassinosteroids. Phytochemistry 37: 1277–1281. 1646–1661. Scheer, J.M., Pearce, G., and Ryan, C.A. (2003). Generation of systemin Li, J.M., and Chory, J. (1997). A putative leucine-rich repeat receptor signaling in tobacco by transformation with the tomato systemin kinase involved in brassinosteroid signal transduction. Cell 90: receptor kinase gene. Proc. Natl. Acad. Sci. USA 100: 10114–10117. 929–938. Scheer, J.M., and Ryan, C.A. (2002). The systemin receptor SR160 Matsubayashi, Y., and Sakagami, Y. (2006). Peptide hormones in from Lycopersicon peruvianum is a member of the LRR receptor plants. Annu. Rev. Plant Biol. 57: 649–674. kinase family. Proc. Natl. Acad. Sci. USA 99: 9585–9590. McGurl, B., Pearce, G., Orozco-Ca´ rdenas, M., and Ryan, C.A. (1992). Schlagnhaufer, C.D., and Arteca, R.N. (1985). Brassinosteroid- Structure, expression, and antisense inhibition of the systemin pre- induced epinasty in tomato plants. Plant Physiol. 78: 300–303. cursor gene. Science 255: 1570–1573. Szekeres, M. (2003). Brassinosteroid and systemin: Two hormones Meindl, T., Boller, T., and Felix, G. (1998). The plant wound hormone perceived by the same receptor. Trends Plant Sci. 8: 102–104. systemin binds with the N-terminal part to its receptor but needs the Szekeres, M., and Bishop, G.J. (2006). Integration of brassinosteroid C-terminal part to activate it. Plant Cell 10: 1561–1570. biosynthesis and signaling. Ann. Plant Rev. 24: 67–92. Montoya, T., Nomura, T., Farrar, K., Kaneta, T., Yokota, T., and Vert, G., Nemhauser, J.L., Geldner, N., Hong, F.X., and Chory, J. Bishop, G.J. (2002). Cloning the tomato curl3 gene highlights the (2005). Molecular mechanisms of steroid hormone signaling in plants. putative dual role of the leucine-rich repeat receptor kinase tBRI1/ Annu. Rev. Cell Dev. Biol. 21: 177–201. SR160 in plant steroid hormone and peptide hormone signaling. Plant Wang, Z.Y., Seto, H., Fujioka, S., Yoshida, S., and Chory, J. (2001). Cell 14: 3163–3176. BRI1 is a critical component of a plasma-membrane receptor for plant Montoya, T., Nomura, T., Yokota, T., Farrar, K., Harrison, K., Jones, steroids. Nature 410: 380–383. J.G.D., Kaneta, T., Kamiya, Y., Szekeres, M., and Bishop, G.J. Yamaguchi, Y., Pearce, G., and Ryan, C.A. (2006). The cell surface (2005). Patterns of Dwarf expression and brassinosteroid accumula- leucine-rich repeat receptor for AtPep1, an endogenous peptide elicitor tion in tomato reveal the importance of brassinosteroid synthesis in Arabidopsis, is functional in transgenic tobacco cells. Proc. Natl. during fruit development. Plant J. 42: 262–269. Acad. Sci. USA 103: 10104–10109. Mussig, C., Shin, G.H., and Altmann, T. (2003). Brassinosteroids Yin, Y.H., Wu, D.Y., and Chory, J. (2002). Plant receptor kinases: promote root growth in Arabidopsis. Plant Physiol. 133: 1261–1271. Systemin receptor identiﬁed. Proc. Natl. Acad. Sci. USA 99: 9090–9092. Tomato BRASSINOSTEROID INSENSITIVE1 Is Required for Systemin-Induced Root Elongation in Solanum pimpinellifolium but Is Not Essential for Wound Signaling Nicholas Holton, Ana Caño-Delgado, Kate Harrison, Teresa Montoya, Joanne Chory and Gerard J. Bishop PLANT CELL 2007;19;1709-1717; originally published online May 18, 2007; DOI: 10.1105/tpc.106.047795

This information is current as of June 23, 2009

Supplemental Data http://www.plantcell.org/cgi/content/full/tpc.106.047795/DC1 References This article cites 35 articles, 21 of which you can access for free at: http://www.plantcell.org/cgi/content/full/19/5/1709#BIBL

Permissions https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw1532298X eTOCs Sign up for eTOCs for THE PLANT CELL at: http://www.plantcell.org/subscriptions/etoc.shtml CiteTrack Alerts Sign up for CiteTrack Alerts for Plant Cell at: http://www.plantcell.org/cgi/alerts/ctmain Subscription Information Subscription information for The Plant Cell and Plant Physiology is available at: http://www.aspb.org/publications/subscriptions.cfm

ADVANCING THE SCIENCE OF PLANT BIOLOGY