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8th International Symposium on Cytochrome P450 Biodiversity and Biotechnology
Dwarfism and cytochrome P450-mediated C-6
oxidation of plant steroid hormones
G. Bishop*1 , T. Nomura†, T. Yokota‡, T. Montoya§, J. Castle§, K. Harrison*, T. Kushiro†, Y. Kamiya†, S. Yamaguchi†,
S. Bancos, A.-M. Szatmári and M. Szekeres
*Division of Biology, Imperial College London, Wye Campus, Kent TN25 5AH, U.K., †Plant Science Center, RIKEN, Institute of Physical and Chemical Research,
Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan, ‡Department of Biosciences, Teikyo University, Utsunomiya 320-8551, Japan, §Institute of Biological
Sciences, University of Wales, Aberystwyth SY23 3DD, U.K., and Institute of Plant Biology, Biological Research Centre of the Hungarian Academy of Sciences,
P.O. Box 521, H-6701 Szeged, Hungary
Abstract
BRs (brassinosteroids) are plant steroid hormones that are essential for normal plant development. The
dramatic dwarfism exhibited by mutants in the CYP (cytochrome P450) enzymes involved in BR biosynthesis
indicates a role for these hormones in plant growth and development. Since the mid-1990s, collaborative
research has been geared towards developing a better understanding of the CYP85 class of CYPs involved
in BR biosynthesis in both Arabidopsis and tomato. Some of the most recent observations include the fact
that certain CYP85 CYPs catalyse the synthesis of the most bioactive BR, BL (brassinolide). Current evidence
suggests that evolution of this function may have occurred independently in different dicotyledonous species.
Interestingly, BL accumulates in tomato fruits, highlighting a key role for this hormone in fruit development.
At the same time as developing a better understanding of the enzymatic function of these CYPs, we have also
carried out experiments towards characterizing where and when these genes are expressed and mechanisms
of their regulation. As expected for a hormone involved in growth and development, biosynthetic gene
promoter activity is associated with young rapidly growing cells and with fruit development.
Background
CYPs (cytochrome P450s) are crucial enzymes required for
hydroxylation/oxidation reactions in both mammalian and
plant steroid hormone synthesis. Since the mid-1990s, rapid
advances have been made in our knowledge and understanding of the biosynthesis and signalling of BRs (brassinosteroids; plant steroid hormones). The key discoveries that
stimulated the BR research were the identification of mutants
involved in BR synthesis and signalling. These mutants provided a role for the most bioactive BR BL (brassinolide) that
had been isolated from rape pollen [1]. The structure of BL
is similar to the mammalian steroid hormones [2]; however,
bioactive BRs are polyhydroxylated on both the A ring and
the side chain (Figure 1).
The importance of the role of BRs in plant growth and
development is easily observed in the striking phenotype of
the dwarf mutants that lack BR synthesis and response. These
mutants are generally dark green and dwarfed in all stages
of plant growth and development. The dwarfism indicates
BR’s involvement in the regulation of growth, photomorphogenesis and fertility. This micro-review will focus on a brief
description of the tomato and Arabidopsis CYP85 CYPs
involved in C-6 oxidation of BRs; please refer to Fujioka
Key words: Arabidopsis, brassinolide, brassinosteroid, cytochrome P450, dwarfism, plant steroid
hormone.
Abbreviations used: BL, brassinolide; BR, brassinosteroid; BR1, brassinosteroid-insensitive 1; CS,
castasterone; CYP, cytochrome P450; 6-deoxoCS, 6-deoxocastasterone.
1
To whom correspondence should be addressed (email [email protected]).
and Yokota [3] or Szekeres and Bishop [4] for more extensive
reviews of BR biosynthesis and metabolism.
CYP85A genes and function
The tomato DWARF gene was isolated via targeted transposon mutagenesis and represented the first member of a
new class of CYPs (CYP85) [5]. The extreme dwarf (dx ) and
transposon-tagged alleles exhibit severe dwarfism having
dark green and crinkled leaves [5]. Yeast that heterologously
expresses CYP85A was fed various BR intermediates as substrates and shown to convert 6-deoxoCS (6-deoxocastasterone-) into CS (castasterone) [6]. This conversion was consistent with the analysis of BR intermediate concentrations in
vegetative tissue of the dx mutant [6]. However, more recent
BR quantification of fruits from the dx mutant and wild-type
indicates the production of BL in both samples [7,8]. A
new CYP85A family gene, named CYP85A3, that is expressed preferentially in tomato fruits was isolated [7]. Functional
expression of CYP85A3 in yeast indicated that this enzyme
catalyses the Baeyer–Villiger oxidation responsible for conversion of CS into BL, in addition to C-6 oxidation of 6deoxoCS to CS [7].
In Arabidopsis, two CYP85A genes, CYP85A1 and
CYP85A2, are present in the genome. The T-DNA (transfer
DNA) insertion lines in CYP85A1 exhibit a normal phenotype but those of CYP85A2 show a weak dwarfism [7,9,10].
The loss-of-function cyp85a1/cyp85a2 double mutant causes
severe dwarfism and sterility, indicating that, unlike tomato,
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Figure 1 CYP85-mediated synthesis of BL
Schematic highlighting the conversion of 6-deoxoCS into BL.
Figure 2 Phylogenetic tree of CYP85 genes
Phylogenetic tree of selected CYP85 full-length protein sequences
R
present in GenBank Nucleotide Sequence Database and rooted using
the CYP90A1 gene from Arabidopsis. The tree was made using MEGA
3.1 program and CLUSTAL W sequence alignment. The tree used the
neighbour-joining method (Bootstrap value, 500) and Poisson correction.
to verify these biological activities using purified enzymes, the
results suggest that either the BL synthase function has independently evolved several times or that this function has been
lost several times. Discerning the function of the rice CYP85
that is present in the genome as a single copy will hopefully clarify the ancestral function of these CYPs.
CYP85A expression
the roles of the two Arabidopsis CYP85A genes are overlapping in growth and development. A functional assay of
the Arabidopsis CYP85A1 in yeast indicated that it catalyses
BR C-6 oxidation in the conversion of 6-deoxoCS into CS
[11]. The Arabidopsis CYP85A2 has recently been shown to
be a BL synthase, similar to the tomato CYP85A3 protein
[7,9]. Both CYP85A1 genes of Arabidopsis and tomato when
expressed in yeast also catalyse the C-6 oxidation of intermediates in the upstream pathway of the BR biosynthesis
[11]. Lines that are defective in both CYP85A1 and CYP85A2
function do not exhibit substantial changes in 6-oxocampestanol levels [10], which indicates that C-6 oxidation of
campestanol is unlikely to be catalysed by a CYP85A enzyme.
CYP85 evolution
There has been differential expansion and loss of CYP families
in plants with the CYP85 family being no exception. Using
selected CYP85 sequences, a phylogeny tree was generated
(Figure 2). This tree indicates a highly novel finding in that
the CYP85s of tomato, pea and Arabidopsis (dicotyledonous
plants) seem to have duplicated independently, whereas in
monocotyledonous plants there is only one gene. This independent duplication also results in the generation of a
novel function, with the CYP85A3 and CYP85A2 genes from
tomato and Arabidopsis encoding the Baeyer–Villiger BL
synthase function. Although further investigation is required
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The transcriptional regulation of CYP85 genes plays a crucial
role in determining BR levels. Bioactive BRs facilitate the
down-regulation of transcript levels of BR-biosynthesis
genes thereby maintaining an optimal level of bioactive BR
production. Such transcriptional feedback regulation of
CYP85 CYPs is the major mechanism involved in altering
BR synthesis. In Arabidopsis, CYP85A1 and CYP85A2 transcripts are down-regulated by bioactive BRs within 2 h of BL
treatment [12–14]. A similar regulation has been seen for the
tomato CYP85A1 gene [8]. The feedback regulation requires
a functional BRI1 (brassinosteroid-insensitive 1) receptor
[8,14–16] because mutants defective in the BRI1 receptor lack
feedback regulation that results in very high CYP transcript
level and BR accumulation [8,17,18].
In addition to the feedback control, organ-specific variation in transcript levels is observed [7,8,12,14]. The spatial
regulation of gene expression provides a mechanism of altering the BR levels in different organs. The CYP85A2 transcripts are at higher levels in shoots than in roots of Arabidopsis
seedlings [14,19]. This is consistent with the fact that 6deoxoCS and CS are at relatively higher concentrations in
Arabidopsis and tomato shoots compared with those in roots,
which have increased levels of 6-deoxotyphasterol and BR
intermediates upstream in the pathway [14,20]. As discussed
above, tomato fruits have been shown to exhibit preferential
expression of CYP85A3 and accumulate BL, highlighting a
key function of BL in fruit development. These localized gene
expression profiles coupled with the lack of BR transport,
as indicated by sectored plants and grafting experiments
[5,21,22], suggest that the sites of expression coincide with the
sites of active BR synthesis [7,22,23]. More recent work has
shown that Arabidopsis CYP85A2 and CYP90A1 transcript
levels exhibit a circadian rhythm of regulation [24]. This
regulation leads to increased levels of BL during the day
8th International Symposium on Cytochrome P450 Biodiversity and Biotechnology
and decreased levels at night, with both the light signalling
pathway and the circadian clock influencing the regulation of
the transcript levels.
G.B. thanks the funding agencies, Biotechnology and Biological
Sciences Research Council, Royal Society, Human Frontier Research
Program and the British Council, for support. Thanks also go to all
co-authors and collaborators who have helped make this research
possible.
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Received 21 June 2006
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