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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,
and systemin, inducing systemic wound responses to
attacks by insect pests, are newly recognized plant
hormones that are perceived by plasma membranelocalized leucine-rich repeat receptor kinases. The
recent characterization of the brassinosteroid receptor
BRI1 from tomato revealed that this protein is identical
to the previously isolated SR160 systemin receptor,
strongly suggesting that both brassinosteroid and
systemin signalling use the same surface receptor.
Brassinosteroids (BRs) are polyhydroxy-steroid phytohormones controlling important developmental functions,
such as growth, photomorphogenesis, fertility, seed germination, senescence and stress tolerance [1]. Brassinolide
(BL), the most active BR, was identified in 1979; several key
elements of its signalling pathway have now been characterized with the help of BR-response mutants. Perception of
BL by BRASSINOSTEROID INSENSITIVE 1 (BRI1), a
plasma membrane-localized leucine-rich repeat (LRR) receptor kinase, probably initiates a phosphorylation cascade that
deactivates the cytoplasmic GSK3/SHAGGY-like BIN2
kinase, a negative regulator of BR signalling. By phosphorylating BES1 and BZR1, two downstream components of
the pathway, BIN2 can prevent their translocation to the
Corresponding author: Miklos Szekeres ([email protected]).
http://plants.trends.com
nucleus, where they act as positive regulators of
BR-responsive gene expression [2– 4].
Considerable attention has been focused on the functional characterization of BRI1, the putative BR receptor.
A series of elegant experiments revealed that the extracellular portion of the protein is required for BR-dependent
activation of the intracellular, Ser/Thr-specific kinase
domain [5,6]. Using radiolabelled BL, it was shown that
BRI1 co-immunoprecipitated with BL binding activity, but
hormone binding was prevented by mutations affecting
the extracellular domain of the protein [7]. These data
unequivocally demonstrated that BRI1 is an essential
component of the BR receptor complex.
Recent cloning and sequence analysis of the tomato
BRI1 gene (tBRI1) by Teresa Montoya and colleagues led
to the exciting discovery that it encodes the Lycopersicon
esculentum equivalent of the Lycopersicon peruvianum
SR160 LRR receptor kinase [8], which had been identified
as the receptor of the peptide hormone systemin [9].
Abs1 and Cu3 encode the tomato ortholog of BRI1
The BR-insensitive mutants of tomato, altered brassinolide sensitivity 1 (abs1, from L. esculentum) [8] and curl 3
(cu3, from Lycopersicon pimpinellifolium) [10] have similar phenotypic features, but abs1 is less dwarfed, develops
an elongated root and retains its fertility and partial
responsiveness to BL (Fig. 1). In both abs1 and cu3,
reverse-transcription PCR assays detected increased
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103
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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%
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Wild type
abs1
–BL
+BL
–BL
+BL
Fig. 1. Partial brassinosteroid-insensitive phenotype of the tomato (Lycopersicon
esculentum) abs1 mutant. Three-week old abs1 and wild-type seedlings were
grown on medium without (þBL) or with (þBL) 1026 M brassinolide. Brassinolide
promotes hypocotyl elongation and retards root development in wild-type
plants but causes only mild root growth inhibition in the abs1 mutant. Scale
bar ¼ 50 mm.
transcript levels of the BR-downregulated Dwarf (BR C-6
oxidase) gene. Furthermore, gas-chromatography massspectrometry analyses showed the accumulation of endogenous BRs, a characteristic feature of the BR-insensitive
mutants [11,12]. Intriguingly, in spite of the nearly 30-fold
accumulation of castasterone, its immediate precursor,
BL could not be detected in the mutant samples. This
seems to confirm the view that castasterone is a bioactive
BR in tomato [13].
Allelism tests between the two mutants clarified that
abs1 is a new, weaker allele of cu3 [8]. Because BRI1 is
thought to be the major non-redundant component of BR
signalling in Arabidopsis, cu3 and cu3 2abs plants were
assumed to carry mutations in the gene encoding tBRI1.
Using an approach that should be useful for isolating
BRI1 homologs from various plant species, a segment of
tBRI1 was PCR-amplified from genomic DNA samples with
degenerate primers corresponding to kinase domain regions
conserved in Arabidopsis and rice BRI1. Using the sequence
information obtained from the PCR product, the complete
tBRI1 gene could then be isolated by inverse PCR.
Sequence analysis of the tBRI1 copy amplified from cu3
revealed a nonsense mutation (G749Z) in the 25th LRR
motif, whereas the tBRI1 amplified from cu3 2abs identified a missense mutation (H1012Y) within the kinase
domain. Co-segregation of the mutation-specific RFLP
patterns with the respective BR-insensitive phenotypes
confirmed that cu3 and cu3 2abs are deficient in the tomato
ortholog of BRI1.
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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 sativum) [8]. The blue bars corresponding to the indicated BRI1 regions show the percentage 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
extracellular (sensor) part with 25 LRR motifs, interrupted by a 70-amino-acid island between the 21st and
22nd LRRs, is connected to the intracellular kinase
domain through a transmembrane segment [14]. Based
on the common role in BR perception, preferential conservation of the functionally important extracellular
regions was expected between the BRI1 proteins of different plant species. Therefore comparing the Arabidopsis
[14], rice [15] and the recently available tomato and pea
BRI1 sequences offered valuable information for identifying potential ligand-binding structures. Pair-wise alignments of these sequences in seven BRI1 regions (Fig. 2)
revealed the highest average amino acid identity between
the kinase domains (83%). Somewhat surprisingly, among
the extracellular regions, the island domain (in which the
known Arabidopsis bri1 mutations are over-represented)
was less conserved (61%) than the flanking LRRs on its
N-terminal (72%) and C-terminal (62%) sides. These data
suggest that the LRRs around the island are crucial for
establishing interaction with the ligand or components of
the BR receptor complex.
Unexpectedly, the amino acid sequence analyses led to
the recognition that tBRI1 is almost fully (. 99%) identical
with the recently identified SR160 systemin receptor [9,16].
Because both LRR receptor kinases are encoded by singlecopy genes and their differences seem to result from interspecific variability (tBRI1 is from L. esculentum whereas
SR160 is from L. peruvianum), these proteins can be
regarded as each other’s functional equivalents.
Tomato BRI1 can function as a dual ligand receptor
Systemin is a Solanaceae family-specific peptide hormone
which, following its release upon wounding, initiates
systemic wound responses, partly through the induction
of jasmonate synthesis [17]. Isolation of the systemin
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receptor was based on its high affinity toward its ligand.
From a cell suspension culture that was photoaffinitylabelled with radioactive systemin, a 160-kDa plasma
membrane protein was purified to homogeneity and, using
its amino acid sequence, identified as the SR160 LRR
receptor kinase [9]. The data presented by Montoya et al.
[8] now suggest that SR160, as well as its BRI1 homolog
from L. esculentum, participate in both BR and systemin
signalling. This result is particularly interesting because
to date only the oxytocin/progesterone receptor of mammals has been known to interact with more than one type
of hormone ligand [18].
How can two phytohormones with such different
structures and physiological functions be perceived by
tBRI1/SR160? The binding of systemin to the receptor
agrees with the anticipated role of LRRs in protein –
protein interactions. By contrast, how BRs bind to the
receptor is unclear. Even at high (1 mM ) concentration, BL
does not compete with systemin in ligand-binding assays,
indicating that BRs and systemin are perceived by different regions of the receptor, or by different binding
mechanisms. For instance, it seems possible that BRI1
perceives BRs as complex ligands formed with sterolbinding proteins [19]. In such a case, this complex could
compete with systemin for the receptor ligand-binding site.
The questions regarding the ligand-specificity of
tBRI1/SR160 remain to be elucidated by future experiments. Like other LRR receptor kinases, BRI1 is expected
to function in a dimeric form: in a yeast expression system,
Arabidopsis BRI1 was shown to form heterodimers and
induce transphosphorylation with the structurally related
Arabidopsis BAK1 receptor kinase [20,21]. Detailed molecular analyses should reveal whether BRI1-interacting
kinase(s) can influence the ligand binding and kinase
specificity of the receptor complex. Furthermore, studying
systemin binding and signalling in BRI1-deficient tomato
mutants, such as cu3 and cu3 2abs, should be instrumental
in clarifying how tBRI1 can participate in both BR and
systemin perception, and how tBRI1 can selectively activate
the respective signalling pathways.
Acknowledgements
I thank Gerard Bishop for the images of tomato seedlings, as well as Éva
Ádám, László Kozma-Bognár and Ferenc Nagy for helpful comments on
the manuscript. Work in my laboratory is supported by the Hungarian
Scientific Research Fund (Grant T 42639).
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1360-1385/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S1360-1385(03)00010-4