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correlation with expression. Nat. Genet. 27, 167–171 1360-1385/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved. 18 Wasserman, W.W. and Fickett, J.W. (1998) Identification of regulatory doi:10.1016/S1360-1385(03)00006-2

Brassinosteroid and systemin: two hormones perceived by the same receptor

Miklos Szekeres

Institute of Plant Biology, Biological Research Centre of the Hungarian Academy of Sciences, POB 521, H-6701 Szeged, Hungary

Brassinosteroids, coordinating developmental events, nucleus, where they act as positive regulators of and systemin, inducing systemic wound responses to BR-responsive gene expression [2–4]. attacks by insect pests, are newly recognized plant Considerable attention has been focused on the func- hormones that are perceived by plasma membrane- tional characterization of BRI1, the putative BR receptor. localized leucine-rich repeat receptor kinases. The A series of elegant experiments revealed that the extra- recent characterization of the brassinosteroid receptor cellular portion of the protein is required for BR-dependent BRI1 from tomato revealed that this protein is identical activation of the intracellular, Ser/Thr-specific kinase to the previously isolated SR160 systemin receptor, domain [5,6]. Using radiolabelled BL, it was shown that strongly suggesting that both brassinosteroid and BRI1 co-immunoprecipitated with BL binding activity, but systemin signalling use the same surface receptor. hormone binding was prevented by mutations affecting the extracellular domain of the protein [7]. These data Brassinosteroids (BRs) are polyhydroxy-steroid phyto- unequivocally demonstrated that BRI1 is an essential hormones controlling important developmental functions, component of the BR receptor complex. such as growth, photomorphogenesis, fertility, seed ger- Recent cloning and sequence analysis of the tomato mination, senescence and stress tolerance [1].Brassinolide BRI1 gene (tBRI1) by Teresa Montoya and colleagues led (BL), the most active BR, was identified in 1979; several key to the exciting discovery that it encodes the Lycopersicon elements of its signalling pathway have now been charac- esculentum equivalent of the Lycopersicon peruvianum terized with the help of BR-response mutants. Perception of SR160 LRR receptor kinase [8], which had been identified BL by BRASSINOSTEROID INSENSITIVE 1 (BRI1), a as the receptor of the peptide hormone systemin [9]. plasma membrane-localized leucine-rich repeat (LRR) recep- tor kinase, probably initiates a phosphorylation cascade that Abs1 and Cu3 encode the tomato ortholog of BRI1 deactivates the cytoplasmic GSK3/SHAGGY-like BIN2 The BR-insensitive mutants of tomato, altered brassino- lide sensitivity 1 (abs1, from L. esculentum) [8] and curl 3 kinase, a negative regulator of BR signalling. By phosphor- (cu3, from Lycopersicon pimpinellifolium) [10] have simi- ylating BES1 and BZR1, two downstream components of lar phenotypic features, but abs1 is less dwarfed, develops the pathway, BIN2 can prevent their translocation to the an elongated root and retains its fertility and partial responsiveness to BL (Fig. 1). In both abs1 and cu3, Corresponding author: Miklos Szekeres ([email protected]). reverse-transcription PCR assays detected increased http://plants.trends.com Update TRENDS in Plant Science Vol.8 No.3 March 2003 103

N-terminal region

15 N-terminal LRRs

6 N-terminal LRRs flanking the island Island domain 4 C-terminal LRRs flanking the island Transmembrane region

Kinase domain

0 25 50 75 100%

TRENDS in Plant Science

abs1 Wild type Fig. 2. Sequence conservation of structural regions between BRI1 proteins (with accession numbers) from Arabidopsis thaliana (AAC49810), rice (Oryza sativa; AP003453.3), tomato (Lycopersicon esculentum; AY179606) and pea (Pisum sati- ÐBL+BL ÐBL +BL vum) [8]. The blue bars corresponding to the indicated BRI1 regions show the per- centage of amino acid sequence identity, calculated as the average of the identity levels obtained by pair-wise alignments using the PileUp program. Abbreviation: LRR, leucine-rich repeat. Sequence conservation between BRI1 orthologs reveals structural requirements of BR perception BRI1 is a typical plasma membrane-localized receptor kinase with complex domain structure. The characteristic Fig. 1. Partial brassinosteroid-insensitive phenotype of the tomato (Lycopersicon extracellular (sensor) part with 25 LRR motifs, inter- esculentum) abs1 mutant. Three-week old abs1 and wild-type seedlings were rupted by a 70-amino-acid island between the 21st and 26 grown on medium without (þBL) or with (þBL) 10 M brassinolide. Brassinolide promotes hypocotyl elongation and retards root development in wild-type 22nd LRRs, is connected to the intracellular kinase plants but causes only mild root growth inhibition in the abs1 mutant. Scale domain through a transmembrane segment [14]. Based bar ¼ 50 mm. on the common role in BR perception, preferential con- transcript levels of the BR-downregulated Dwarf (BR C-6 servation of the functionally important extracellular oxidase) gene. Furthermore, gas-chromatography mass- regions was expected between the BRI1 proteins of dif- ferent plant species. Therefore comparing the Arabidopsis spectrometry analyses showed the accumulation of endo- [14], rice [15] and the recently available tomato and pea genous BRs, a characteristic feature of the BR-insensitive BRI1 sequences offered valuable information for identify- mutants [11,12]. Intriguingly, in spite of the nearly 30-fold ing potential ligand-binding structures. Pair-wise align- accumulation of castasterone, its immediate precursor, ments of these sequences in seven BRI1 regions (Fig. 2) BL could not be detected in the mutant samples. This revealed the highest average amino acid identity between seems to confirm the view that castasterone is a bioactive the kinase domains (83%). Somewhat surprisingly, among BR in tomato [13]. the extracellular regions, the island domain (in which the Allelism tests between the two mutants clarified that known Arabidopsis bri1 mutations are over-represented) abs1 is a new, weaker allele of cu3 [8]. Because BRI1 is was less conserved (61%) than the flanking LRRs on its thought to be the major non-redundant component of BR 2abs N-terminal (72%) and C-terminal (62%) sides. These data signalling in Arabidopsis, cu3 and cu3 plants were suggest that the LRRs around the island are crucial for assumed to carry mutations in the gene encoding tBRI1. establishing interaction with the ligand or components of Using an approach that should be useful for isolating the BR receptor complex. BRI1 homologs from various plant species, a segment of Unexpectedly, the amino acid sequence analyses led to tBRI1 was PCR-amplified from genomic DNA samples with the recognition that tBRI1 is almost fully (.99%) identical degenerate primers corresponding to kinase domain regions with the recently identified SR160 systemin receptor [9,16]. conserved in Arabidopsis and rice BRI1. Using the sequence Because both LRR receptor kinases are encoded by single- information obtained from the PCR product, the complete copy genes and their differences seem to result from inter- tBRI1 gene could then be isolated by inverse PCR. specific variability (tBRI1 is from L. esculentum whereas Sequence analysis of the tBRI1 copy amplified from cu3 SR160 is from L. peruvianum), these proteins can be revealed a nonsense mutation (G749Z) in the 25th LRR regarded as each other’s functional equivalents. motif, whereas the tBRI1 amplified from cu3 2abs identi- fied a missense mutation (H1012Y) within the kinase Tomato BRI1 can function as a dual ligand receptor domain. Co-segregation of the mutation-specific RFLP Systemin is a Solanaceae family-specific peptide hormone patterns with the respective BR-insensitive phenotypes which, following its release upon wounding, initiates confirmed that cu3 and cu3 2abs are deficient in the tomato systemic wound responses, partly through the induction ortholog of BRI1. of jasmonate synthesis [17]. Isolation of the systemin http://plants.trends.com 104 Update TRENDS in Plant Science Vol.8 No.3 March 2003 receptor was based on its high affinity toward its ligand. metabolism, transport, perception and response. Plant Cell Physiol. From a cell suspension culture that was photoaffinity- 42, 114–120 labelled with radioactive systemin, a 160-kDa plasma 2 Clouse, S.D. (2002) Brassinosteroid signal transduction. Clarifying the pathway from ligand perception to gene expression. Mol. Cell 10, membrane protein was purified to homogeneity and, using 973–982 its amino acid sequence, identified as the SR160 LRR 3 Friedrichsen, D. and Chory, J. (2001) Steroid signaling in plants: from receptor kinase [9]. The data presented by Montoya et al. the cell surface to the nucleus. BioEssays 23, 1028–1036 [8] now suggest that SR160, as well as its BRI1 homolog 4 Zhao, J. et al. (2002) Two putative BIN2 substrates are nuclear components of brassinosteroid signaling. Plant Physiol. 130, from L. esculentum, participate in both BR and systemin 1221–1229 signalling. This result is particularly interesting because 5 He, Z.H. et al. (2000) Perception of brassinosteroids by the extracellu- to date only the oxytocin/progesterone receptor of mam- lar domain of the receptor kinase BRI1. Science 288, 2360–2363 mals has been known to interact with more than one type 6 Oh, M.H. et al. (2000) Recombinant brassinosteroid insensitive 1 of hormone ligand [18]. receptor-like kinase autophosphorylates on serine and threonine residues and phosphorylates a conserved peptide motif in vitro. How can two phytohormones with such different Plant Physiol. 124, 751–765 structures and physiological functions be perceived by 7 Wang, Z-Y. et al. (2001) BRI1 is a critical component of a plasma- tBRI1/SR160? The binding of systemin to the receptor membrane receptor for plant steroids. Nature 410, 380–383 agrees with the anticipated role of LRRs in protein– 8 Montoya, T. et al. (2002) Cloning the tomato Curl3 gene highlights protein interactions. By contrast, how BRs bind to the the putative dual role of the leucine-rich repeat receptor kinase tBRI1/SR160 in plant steroid hormone and peptide hormone signaling. receptor is unclear. Even at high (1 mM) concentration, BL Plant Cell 14, 3163–3176 does not compete with systemin in ligand-binding assays, 9 Scheer, J.M. and Ryan, C.A. Jr (2002) The systemin receptor SR160 indicating that BRs and systemin are perceived by dif- from Lycopersicon peruvianum is a member of the LRR receptor kinase ferent regions of the receptor, or by different binding family. Proc. Natl. Acad. Sci. U. S. A. 99, 9585–9590 mechanisms. For instance, it seems possible that BRI1 10 Koka, C.V. et al. (2000) A putative role for the tomato genes DUMPY and CURL3 in brassinosteroid biosynthesis and response. Plant perceives BRs as complex ligands formed with sterol- Physiol. 122, 85–98 binding proteins [19]. In such a case, this complex could 11 Nomura, T. et al. (1997) Blockage of brassinosteroid biosynthesis compete with systemin for the receptor ligand-binding site. and sensitivity causes dwarfism in garden pea. Plant Physiol. 113, The questions regarding the ligand-specificity of 31–37 tBRI1/SR160 remain to be elucidated by future experi- 12 Noguchi, T. et al. (1999) Brassinosteroid-insensitive dwarf mutants of Arabidopsis accumulate brassinosteroids. Plant Physiol. 121, 743–752 ments. Like other LRR receptor kinases, BRI1 is expected 13 Nomura, T. et al. (2001) Accumulation of 6-deoxocathasterone and to function in a dimeric form: in a yeast expression system, 6-deoxocastasterone in Arabidopsis, pea and tomato is suggestive of Arabidopsis BRI1 was shown to form heterodimers and common rate-limiting steps in brassinosteroid biosynthesis. Phyto- induce transphosphorylation with the structurally related chemistry 57, 171–178 Arabidopsis BAK1 receptor kinase [20,21]. Detailed mol- 14 Li, J. and Chory, J. (1997) A putative leucine-rich repeat receptor kinase involved in brassinosteroid signal transduction. Cell 90, ecular analyses should reveal whether BRI1-interacting 929–938 kinase(s) can influence the ligand binding and kinase 15 Yamamuro, C. et al. (2000) Loss of function of a rice brassinosteroid specificity of the receptor complex. Furthermore, studying insensitive 1 homolog prevents internode elongation and bending of systemin binding and signalling in BRI1-deficient tomato the lamina joint. Plant Cell 12, 1591–1605 mutants, such as cu3 and cu3 2abs, should be instrumental 16 Yin, Y. et al. (2002) Plant receptor kinases: systemin receptor identified. Proc. Natl. Acad. Sci. U. S. A. 99, 9090–9092 in clarifying how tBRI1 can participate in both BR and 17 Lindsey, K. et al. (2002) Peptides: new signalling molecules in plants. systemin perception, and how tBRI1 can selectively activate Trends Plant Sci. 7, 78–83 the respective signalling pathways. 18 Grazzini, E. et al. (1998) Inhibition of oxytocin receptor function by direct binding of progesterone. Nature 392, 509–512 Acknowledgements 19 Bishop, G.J. and Koncz, C. (2002) Brassinosteroids and plant steroid hormone signaling. Plant Cell 14, S97–S110 I thank Gerard Bishop for the images of tomato seedlings, as well as E´ va 20 Nam, K.H. and Li, J. (2002) BRI1/BAK1, a receptor kinase pair A´ da´m, La´szlo´ Kozma-Bogna´r and Ferenc Nagy for helpful comments on mediating brassinosteroid signaling. Cell 110, 203–212 the manuscript. Work in my laboratory is supported by the Hungarian 21 Li, J. et al. (2002) BAK1, an Arabidopsis LRR receptor-like protein Scientific Research Fund (Grant T 42639). kinase, interacts with BRI1 and modulates brassinosteroid signaling. Cell 110, 213–222 References

1 Bishop, G.J. and Yokota, T. (2001) Plants steroid hormones, brassi- 1360-1385/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved. nosteroids: Current highlights of molecular aspects on their synthesis/ doi:10.1016/S1360-1385(03)00010-4

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