Note: Descriptions are shown in the official language in which they were submitted.
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DWF4 POLYNUCLEOTIDES, POLYPEPTIDES AND USES THEREOF
TECHNICAL FIELD
The present invention relates to novel polynucleotides isolated from dwarf
plants. The dw, f4 polynucleotides encode all, or a portion of, a DWF4
polypeptide, a
cytochrome P450 enzyme that mediates multiple steps in synthesis of
brassinosteroids. The present invention also relates to isolated
polynucleotides that
encode regulatory regions of dwf4. Uses of the dwf4 polypeptides and
polynucleotides are also disclosed.
BACKGROUND
Plant growth is accomplished by orderly cell division and tightly regulated
cell
expansion. In plants, the contribution of cell expansion to growth is of much
greater
significance than in most other organisms; all plant organs owe their final
size to a
period of significant cell elongation, which usually follows active cell
division.
Further, the sessile nature of plants requires that they make fine but
responsive
adjustments in growth to survive harsh environmental conditions and to
optimize their
use of limited resources (Trewavas (1986) "Resource allocation under poor
growth
conditions: A major role for growth substances in developmental plasticity" In
Plasticity in Plants, D.H. Jennings and A.J. Trewavas, eds (Cambridge, UK:
Company
of Biologists Ltd.), pp. 31-76).
In Arabidopsis, cell elongation is largely responsible for hypocotyl growth in
germinating seedlings and extension of inflorescences (bolting) at the end of
vegetative growth. Coordinate control of plant growth is regulated by both
external
stimuli and internal mechanisms. Of the external signals, the most obvious is
light
(Deng, X.-W. (1994) Cell 76:423-426). Light inhibits hypocotyl elongation and
promotes cotyledon expansion and leaf development in seedlings, and
photoperiod is
crucial for flower initiation in a large number of species.
The internal components of plant signaling are generally mediated by
chemical growth regulators (phytohormones; reviewed in Klee, H., and Estelle,
M.
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(1991 ) Annu. Rev. Plant Physiol. Plant Mol. Biol. 42:529-551 ). Thus, plant
growth
in response to environmental factors is modulated by plant hormones acting
alone or
in concert (Evans "Functions of hormones at the cellular level of
organization" In
Hormonal Regulation of Plant Physiology, T.K. Scott, ed (Berlin: Springer-
Verlag),
pp. 23-79), and growth depends on regulated cellular events, such as division,
elongation, and differentiation.
Gibberellic acid (GA) and cytokinins promote flowering; in addition, GA
stimulates stem elongation, whereas cytokinins have the opposite effect,
reducing
apical dominance by stimulating increased axillary shoot formation.
Conversely,
auxins promote apical dominance and stimulate elongation by a process
postulated to
require acidification of the cell wall by a K--dependent H+-pumping ATPase
(Rayle,
D.L., and Cleland, R.E. ( 1977) Curr. Top. Dev. Biol. 11:187-214).
In addition to the classic hormones, such as auxin and gibberellic acid (GA),
brassinosteroids (BRs) have been discovered to be important in growth
promotion
(reviewed in Clouse (1996) Plant J. 10:1-8). The most recently discovered
class of
plant growth substances, the BRs, has been to date the least studied; however,
rapid
progress toward understanding BR biosynthesis and regulation is now being made
(Yokota, T. (1997) Trends Plant Sci. 2:137-143). The term BRs collectively
refers to
the growth-promoting steroids found in plants (Grove et al. (1979) Nature
281:216-217). They are structurally very similar to the molting hormones of
insects,
ecdysteroids (Richter and Koolman ( 1991 ) "Antiecdysteroid effects of
brassinosteroids in insects" in Brassinosteroids: Chemistry, Bioactivity, and
Applications, H.G. Cutler, T. Yokota, and G. Adam, eds (Washington, DC:
American
Chemical Society), pp. 265-279), but active BRs have unique structural
features. As
shown in Figure 1, a 6-oxolactone or 7-oxalactone in the B ring, 5a hydrogen,
and
multiple hydroxylations at four different positions with specific
stereochemistry have
been proposed as an essential configuration for BRs (reviewed in Marquardt and
Adam (1991) "Recent advances in brassinosteroid research" in Chemistry of
Plant
Protection, W. Ebing, ed (Berlin: Springer-Verlag), pp. 103-139). Among >40
naturally occurnng BRs, brassinolide (BL; 2a, 3a, 22(R), 23(R)-tetrahydroxy-
24(S)-
methyl-B-homo-7-oxa-5a-cholestan-6-one) has been shown to be the most
biologically active (reviewed in Mandava (1988) Annu. Rev. Plant Physiol.
Plant
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Mol. Biol. 39:23-52 ). As a major biological effect, BRs stimulate
longitudinal
growth of young tissues via cell elongation and cell division (reviewed in
Clouse
(1996), supra; Fujioka and Sakurai (1997a) Nat. Prod. Rep. 14:1-10).
Elucidating the BR biosynthetic pathways has been a major area of recent
interest. Biochemical analyses have been used to elucidate the BR biosynthetic
pathway (Fujioka et al. (1996) Plant Cell Physiol. 37:1201-1203; Choi et al.
(1997),
Phytochemistry 44:609-613), and mutational analyses are being used to confirm
this
pathway. Similar to the biosynthetic pathways of the human steroid hormones
and
insect ecdysteroids (Rees (1985) "Biosynthesis of ecdysone" in Comprehensive
Insect
Physiology, Biochemistry and Pharmacology, G.A. Kerkut and L.I. Gilbert, eds
(Oxford, UK: Pergamon Press), pp. 249-293; Granner, D.K. (1996) "Hormones of
the
gonads" in Harper's Biochemistry, R.K. Murray, D.K. Granner, P.A. Mayes, and
V.W. Rodwell, eds (Stamford, CT: Appleton and Lange Press), pp. 566-580), BRs
are
synthesized via multiple parallel pathways (Fujioka et al. (1996) Plant Cell
Physiol.
37:1201-1203; Choi et al. (1997), supra). Starting from the initial precursor,
campesterol (CR), the BR intermediates undergo a series of hydroxylations,
reductions, an epimerization, and a Baeyer-Villigerutype oxidation leading to
the
most oxidized form, BL (Fujioka and Sakurai (1997b) Physiol. Plant. 100:710-
715;
Figure 1). Castasterone (CS) oxidation, the last step in BR biosynthesis, is
not found
in some species, such as mung bean. In that case, CS plays a role as the major
BR
rather than BL (Yokota et al. (1991) "Metabolism and biosynthesis of
brassinosteroids" in Brassinosteroids: Chemistry, Bioactivity, and
Application, H.G.
Cutler, T. Yokota, and G. Adam, eds (Washington, DC: American Chemical
Society),
pp. 86-96). Traditionally, BR biosynthetic pathways have been elucidated by
feeding
deuterio-labeled intermediates to BR-producing cell lines of Madagascar
periwinkle
(Sakurai and Fujioka (1996) "Catharanthus roseus (Vinca rosea): In vitro
production
of brassinosteroids" in Biotechnology in Agriculture and Forestry, Y.P.S.
Bajaj, ed
(Berlin: Springer-Verlag), pp. 87-96.). The present model, including parallel
branched pathways and early and late C-6 oxidation pathways, was established
using
these feeding studies (Fujioka and Sakurai (1997a), supra, Fujioka and Sakurai
(1997b), supra; Sakurai and Fujioka (1997) Biosci. Biotechnol. Biochem.
61:75 7-762).
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Although the brassinosteriod system is a less well understood class of plant
growth substances (BRs; Mitchell, et al. (1970) Nature 225:1065-1066; Grove et
al.
(1979) Nature 281:216-217; Mandava, N.B. (1988) Annu. Rev. Plant Physiol.
Plant
Mol. Biol. 39:23-52), several such compounds have been identified and are
known to
effect elongation of cells in various plant tissues, their biosynthesis,
regulation, and
mechanism of action have only recently begun to be elucidated (reviewed in
Clouse,
S.D. (1996) Plant J. 10:1-8; Fujioka, S., and Sakurai, A. (1997) Physiol.
Plant.
100:710-715).
Several types of dwarf or dwarflike mutants have been described in
Arabidopsis. A number of mutations have been identified that affect either
light-dependent (cop, det, and fusca [fus; another group of mutants with some
members perturbed in light-regulated growth]) or hormone signaling (axr2)
pathways
and whose pleiotropic phenotypes include defects in cell elongation. The
majority of
these mutants also have other alterations in their phenotypes. At least five
GA
mutants have been described as being reduced in stature (Koornneef and Van der
Veen (1980) Theor. Appl. Genet. 58:257-263). GA biosynthetic mutants may also
have no or defective flower development and are marked by an absence of viable
pollen. Reduced levels of endogenous gibberellins are also a characteristic
(Barendse
et al.(1986) Physiol. Plant. 67:315-319; Talon et al. (1990) Proc. Natl. Acad.
Sci.
USA 87:7983-7987), and their phenotype can be nearly restored to that of the
wild
type by the addition of exogenous GA. (Koornneef and Van der Veen (1980)
Theor.
Appl. Genet. 58:257-263).
Another hormone mutation, auxin resistant2 (axr2), results in plants with a
dwarf phenotype both in the light and in darkness as well as increased
resistance to
high levels of auxin, ethylene, and abscisic acid (Timpte et al. (1992) Planta
188:271-278). An interesting relationship exists between light regulation and
cytokinin levels. Arabidopsis seedlings grown in the dark in the presence of
cytokinins have open cotyledons, initiate chloroplast differentiation and leaf
development, and activate transcription from the chlorophyll alb binding
protein gene
(CAB) promoter. Importantly, they also display a cytokinin dose-dependent
dwarf
phenotype.
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Dwarf Arabidopsis mutants that are rescued by addition of BRs have also been
described (Kauschmann et al. (1996) Plant J. 9:701-713; Li et al. (1996)
Science
272:398-401; Szekeres et al. (1996) Cell 85:171-182; Azpiroz et al. (1998)
Plant Cell
10:219-230), including the following three mutants: dwarfl (dwfl ; Kauschmann
et al.
(1996) Plant J. 9:701-713), constitutive photomorphogenesis and dwarfism (cpd;
Szekeres et al. (1996) Cell 85:171-182), and det2 (Li et al. (1996) Science
272:398-401). These mutants have been shown to be defective in steroid
biosynthesis. DWFI (Feldmann et al. (1989) Science 243:1351-1354) was cloned
first (GenBank accession number U12400). Takahashi et al. (1995) Genes Dev.
9:97-107 hypothesized that DWF1, which they isolated with an allele of dwfl,
referred to as diminuto 1 (diml ), contains a potential nuclear targeting
signal, which
may confer a regulatory function to the protein. However, Mushegian and Koonin
(1995) Protein Sci. 4:1243-1244 indicated that DWF1 displays limited homology
with
flavin adenine dinucleotide (FAD)independent oxidoreductase, suggesting an
enzymatic function in BR biosynthesis. According to Kauschmann et al. (1996),
supra (dwfl-6 described as cabbagel [cbbl]), dwfl mutants were rescued by
exogenous application of BRs.
DET2 was shown to encode a putative steroid Sa-reductase, mediating an
early step in BR biosynthesis (Li et al. (1996), supra , Li et al. (1997)
Proc. Natl.
Acad. Sci. USA 94:3554-3559; Fujioka et al. (1997) Plant Cell 9:1951-1962;
Figure
1 ). Moreover, detl and det2 have a decreased requirement for cytokinins in
tissue
culture and appear to be saturated for a cytokinin-dependent delay in
senescence
(Chory et al. (1994) Plant Physiol. 104:339-347). CPD has been proposed to be
a
novel cytochrome P450 (CYP90A1; Szekeres et al. (1996), supra), encoding a
putative 23a-hydroxylase that acts in BR biosynthesis. The range of phenotypes
in
the deetiolated (det) and constitutive photomorphogenic (cop) light-regulatory
mutants is broad. Mutations in DETI , COPI , COPB, COP9, COPI 0, and COPT I
result in constitutive derepression of substantial portions of the
photomorphogenic
program (Chory, et al. (1989b) Cell 58:991-999; Deng, X.-W., and Quail, P.H.
(1992)
Plant J. 2:83-95; Wei, N., and Deng, X.-W. (1992) Plant Cell 4:1507-1518; Wei
et al.
(1994) Plant Cell 6:629-643), whereas mutations in COP4 seem to affect only
morphology and gene expression (Hou et al. (1993) Plant Cell 5:329-339). The
only
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invariant phenotype in this class of light-regulatory mutants is a substantial
reduction
in height in both light and darkness.
There are additional dwarfs that are insensitive to one of these hormones,
such
as bri (brassinosteroid insensitive; Clouse et al. (1996) Plant Physiol.
111:671-678;
Li and Chory (1997) Cell 90:929-938), gai (gibberellic acid insensitive;
Koornneef et
al. (1985) Physiol. Plant. 65:33-39), and axr2 (auxin resistant2; Timpte et
al. (1994)
Genetics 138:1239-1249). Clouse et al. (1996), supra isolated bri by screening
ethyl
methanesulfonate-mutagenized populations for mutants whose root growth is not
retarded at inhibitory concentrations of BR. Thus, the BRI protein is proposed
to be
involved in BR signal perception or transduction (Clouse (1996), supra).
Kauschmann et al. (1996), supra described a phenotypically similar mutant cbb2
that
maps to the same location. In addition, the dwf2 alleles possess a phenotype
similar
to bri and map to the same region (Feldmann and Azpiroz ( 1994) "dwarf (dw~
and
twisted dwarf (twd)" in Arabidopsis: An Atlas of Morphology and Development,
J.
Bowman, ed (New York: Springer-Verlag), pp. 82-85). It seems likely that all
of the
BR-insensitive dwarf mutants described to date are allelic. Recently, BRI has
been
cloned and shown to encode a leucine-rich-repeat receptor kinase, suggesting a
role in
the BR signal transduction pathway (Li and Chory (1997), supra).
Mutants defective in BR biosynthesis have also been isolated in other plant
species. Bishop et al. (1996) Plant Cell 8:959-969 isolated a tomato dwarf
mutant by
transposon tagging. The tomato Dwarf gene encodes a pioneering member of the
CYP85 family, and it appears to be involved in BR biosynthesis. In addition,
Nomura
et al. (1997) Plant Physiol. 113:31-37 reported that the Ika and Ikb mutants
in garden
pea are deficient in BR biosynthesis (lkb) or perception (lka).
Currently, little is known about the downstream events that occur in response
to these signals and thereby directly control cell size. This is because the
biochemical
and cell biological processes involved have thus far been difficult to
address. In
addition, there is little information about the integration of regulatory
signals
converging at the cell from different signaling pathways and the ways they are
coordinately controlled. In particular, the interaction of light and hormones
in the
control of cell elongation is not clear. Thus, there remains a need for the
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identification and characterization of additional mutants and polypeptides
encoded
thereby involved in these pathways of plant growth.
SUMMARY OF THE INVENTION
In one aspect the invention includes an isolated dwf4 polynucleotide
comprising an open reading frame that encodes a polypeptide comprising (i) a
sequence having greater than 43% identity to the amino acid sequence of
SEQUENCE ID N0:2; (ii) a sequence comprising at least about 10 contiguous
amino
acids that have greater than 43% identity to 10 contiguous amino acids of
SEQUENCE ID N0:2, or a complement or reverse complement of said
polynucleotide. In certain embodiments, the polynucleotide will have at least
70%
identity to the DWF4 polypeptide-coding region of SEQ ID NO:1 or to
complements
and reverse complements of this region. In further embodiments, the isolated
dwf4
polynucleotide comprises the nucleotide sequence of SEQ ID NO:1, complements
and
reverse complements thereof. The polynucleotide may also comprise at least 30
consecutive nucleotides of SEQ ID NO: l .
In another aspect, the invention includes an isolated dwf4 polynucleotide
comprising (i) a sequence having at least 50% identity to SEQ ID NO:1,
complements
and reverse complements thereof or (ii) a sequence comprising at least about
15
contiguous nucleotides that has at least SO% identity to SEQ ID NO:1,
complements
and reverse complements thereof. In certain embodiments, the isolated dwf4
polynucleotide has at least 50% identity to the DWF4 polypeptide-coding region
of
SEQ ID NO:1, complements and reverse complements thereof. In further
embodiments, the isolated dwf4 polynucleotides described herein comprise the
nucleotide sequence of SEQ ID NO:1, complements and reverse complements
thereof
or nucleotide sequences comprising at least 30 consecutive nucleotides of SEQ
ID
NO:1. Any of the dwf4 polynucleotides described herein may be genomic DNA and
may include introns. Further, in other embodiments, the dwf4 polynucleotide
includes
a dwf4 control control element comprising a polynucleotide selected from the
group
consisting of (i) a sequence having at least 50% identity to nucleotides 1 to
3202 of
SEQ ID NO:l; (ii) a fragment of (i) which includes a dwf4 control element; and
(iii)
complements and reverse complements of (i) or (ii). In still further
embodiments, the
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polynucleotide includes a dwf4 control element comprising a polynucleotide
selected
from the group consisting of (i) a sequence having at least 50% identity to
nucleotides
6111 to 6468 corresponding to the 3' UTR of SEQ ID NO: l; (ii) a fragment of
(i)
which includes a dwf4 3' UTR; and (iii) complements and reverse complements of
(i)
or (ii). In certain embodiments, the polynucleotide includes a dwf4
polynucleotide
selected from the group consisting of (i) a sequence having at least 50%
identity to the
sequences corresponding to the introns of SEQ ID NO:1; (ii) a fragment of (i)
which
includes a dwf4 intro; and (iii) complements and reverse complements of (i)
and (ii).
Introns are found, for example, in the following regions: nucleotides 3424 to
3503 of
SEQ ID NO:1; nucleotides 3829 to 3913 of SEQ ID NO:l; nucleotides 4067 to 4164
of SEQ ID NO:1; nucleotides 4480 to 4531 of SEQ ID NO:I; nucleotides 4725 to
4815 of SEQ ID NO:1; nucleotides 4895 to 5000 of SEQ ID NO:1; and nucleotides
5111 to 5864 of SEQ ID NO:1. 54. In still further embodiments, any of the
polynucleotides described herein can operably linked to a nucleic acid
molecule
encoding a heterologous polypeptide (e.g., a cytochrome P450 polypeptide), for
example, as a chimeric polynucleotide.
In another aspect, the invention includes recombinant vectors comprising (i)
one or more of the polynucleotides described above; and (ii) control elements
operably linked to the one or more polynucleotides, whereby a coding sequence
within said polynucleotide can be transcribed and translated in a host cell.
In certain
embodiments, the recombinant vector comprises (a) any of the polynucleotides
which
include a dwf4 control element described above (e.g., promoter or intron); and
(b) a
nucleic acid molecule comprising a coding sequence operably linked to the dwf4
control element.
Host cells comprising and/or transformed with any of the recombinant vectors
described herein are also provided. In certain embodiments, the host cells are
cultured
ex vivo while in other embodiments, the dwf4 polynucleotide is provided the
host cell
in vivo. In certain embodiments the DWF4 polypeptide is provided in amounts
such
that a plant is regenerated.
In another aspect, the present invention includes a method of modulating a
DWF4 polypeptide comprising the following steps: (a) providing a host cell as
described herein; and (b) culturing said host cell under conditions whereby
the dwf4
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polynucleotide included in the host cell is transcribed. In certain
embodiments, the
dwf4 polynucleotide is overexpressed. Alternatively, in other embodiments, the
polynucleotide included in the host cell inhibits expression of dwf4.
In yet another aspect, the present invention includes a transgenic plant
comprising any of the recombinant vectors described herein.
In yet another aspect, the invention includes a method of producing a
recombinant polypeptide comprising the following steps: (a) providing a host
cell as
described herein; and (b) culturing said host cell under conditions whereby
the
recombinant polypeptide encoded by the coding sequence present in said
recombinant
vector is expressed.
In a still further aspect, the invention includes a method of producing a
transgenic plant comprising the steps of (a) introducing a polynucleotide
described
herein into a plant cell to produce a transformed plant cell; and (b)
producing a
transgenic plant from the transformed plant cell.
Methods for producing a transgenic plant having an altered phenotype relative
to the wild-type plant comprising the following steps: introducing at least
one
polynucleotide described herein into a plant cell; and producing a transgenic
plant
from the plant cell, said transgenic plant having an altered phenotype
relative to the
wild-type plant are also included in the present invention. The altered
phenotype
includes altered morphological appearance and altered biochemical activity,
for
example, altered (reduced or increased) cell length in any cell or tissue,
altered
(extended or decreased) periods of flowering, altered (increased or decreased)
branching, altered (increased or decreased) seed production, altered
(increased or
decreased) leaf size, altered (elongated or shortened) hypocotyls, altered
(increased or
decreased) plant height, altered heme-thiolate enzyme activity, altered
monooxygenase activity, altered 22a-hydroxylase activity, regulation of
brassinosteriod synthesis, regulation of gibberellic acid, regulation of
cytokinins,
regulation of auxins, altered resistance to plant pathogens, altered growth at
low
temperatures, altered growth in dark conditions and altered sterol
composition. In
certain embodiments, the at least one polynucleotide is operably linked to a
promoter
selected from the group consisting of a tissue-specific promoter, an inducible
promoter or a constitutive promoter. The polynucleotide can be overexpressed
or it
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can inhibit expression of dwf4. In a still further embodiment, at least two
polynucleotides are introduced into the plant cell. Each polynucleotide is
operably
linked to a different tissue-specific promoter such that one polynucleotide is
overexpressed while the other inhibits expression of dwf4.
In yet another aspect, the invention includes a method for altering the
biochemical activity of a cell comprising the following steps: introducing at
least one
polynucleotide described herein; and culturing the cell under conditions such
that the
biochemical activity of the cell is altered. Biochemical activity includes,
for example,
altered heme-thiolate enzyme activity, altered monooxygenase activity, altered
22x-
hydroxylase activity, regulation of gibberellic acid, regulation of
cytokinins,
regulation of auxins, and altered sterol composition. In certain embodiments,
the cell
is cultured ex vivo. In other embodiments, the dwf4 polynucleotide is provided
to the
cell in vivo. In still other embodiments, more than one dwf4 polynucleotides
are
provided to the cell.
1 S In yet another aspect, the invention includes a method for regulating the
cell
cycle of a plant cell comprising the following steps providing a dwf4
polynucleotide
to a plant cell; and expressing the dwf4 polynucleotide to provide a DWF4
polypeptide, wherein the DWF4 polypeptide is provided in amounts such that
cell
cycling is regulated. In certain embodiments, the plant cell is provided in
vitro and is
cultured under conditions suitable for providing the DWF4 polypeptide. In
still other
embodiments, the dwf4 polynucleotide is provided in vivo.
In yet another aspect, the invention includes an isolated DWF4 polypeptide
comprising (i) a sequence having greater than 43% identity to SEQ ID N0:2 or
(ii)
fragments of (i) that confer a DWF4 phenotype when expressed in a host
organism.
In certain embodiments, the isolated DWF4 polypeptide comprises the amino acid
sequence of SEQ ID N0:2. In certain embodiments, the invention includes a
chimeric
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polypeptide comprising a DWF4 polypeptide as described herein and a
heterologous
polypeptide, for example a cytochrome P450 polypeptide.
Any of the polynucleotides or polypeptides described herein can be used in
diagnostic assays; to generate antibodies. Further, the antibodies and
fragments
thereof can also be used in diagnostic assays, to produce immunogenic
compositions
or the like.
These and other objects, aspects, embodiments and advantages of the present
invention will readily occur to those of ordinary skill in the art in view of
the
disclosure herein.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 depicts a proposed biosynthetic pathway for BL. CR goes through at
least two different pathways, referred to as the early C-6 oxidation (right
column) and
late C-6 oxidation (left column) pathways. Steps mediated by DWF4, CPD
(Szerkeres et al. (1996), supra), DET2 (Fujioka and Skaurai (1997a), infra; Li
et al.
(1997), supra) and LKB (Yokota et al. (1997), infra) are indicated.
Figures 2A and B depict schematic representations of the DWF4 gene and
protein. Figure 2A depicts the DWF4 coding sequence (1542 bp) and shows that
the
coding sequence contains eight exons and seven introns. The exons and introns
range
in length from 93 to 604 and 84 to 754 bp, respectively. All of the introns
are
bordered by typical consensus splice junctions, 5'-GU and AG-3'. Closed
rectangles
indicate exons. The T-DNA position in dwf4-1 is marked with an arrow. Figure
2B
shows the relative positions of the major domains in DWF4 cytochrome P450. All
of
the major domains found in the cytochrome P450 superfamily are conserved in
DWF4. The estimated molecular mass and isoelectric point of the DWF4 protein
were 58 kD and 7.28, respectively. Hydropathy plotting and protein
localization
prediction by the PSORT software package (Nakai and Kaneshia (1992) Genomics
14:897-911 ) suggested that the protein may reside in a membrane of the
endoplasmic
reticulum as an integral protein. Mutations identified in the other dwf4
alleles are
indicated.
Figure 3 depicts alignment of cytochrome P450 proteins that exhibited the
most similarity to DWF4 in BLAST searches. GenBank accession numbers are
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AF044216 (DWF4; CYP90B), X87368 (CPD; CYP90A), U54770 (tomato; CYP85),
D64003 (cyanobacteria; CYP120), U32579 (maize; CYP88), U68234 (zebrafish;
CYP26), and M13785 (human; CYP3A3X). Dashes indicate gaps introduced to
maximize alignment. Domains indicated in Figure 2B are highlighted in a box.
Amino
acid residues that are conserved >50% between the compared sequences are
highlighted by a reverse font, and identical residues between DWF4 and CPD are
boxed and italicized. Open triangles are placed under the 100% conserved
residues.
Closed triangles locate functionally important amino acid residues, for
example,
threonine (T ) at 369, which is thought to bind molecular oxygen, and cysteine
(C) at
516, which links to a heme prosthetic group by a thiolate bond. X's indicate
mutated
residues in dwf4 alleles. Multiple sequence alignment was performed using
PILEUP
in the Genetics Computer Group package, and box shading was made possible by
the
ALSCRIPT package (Burton (1993) Protein Eng. 6:37-40).
Figure 4 depicts the phylogenetic Relationship between DWF4 and Selected
Cytochrome P450s. DWF4 did not cluster with the group A plant cytochrome P450s
that are known to mediate plant-specific reactions (Durst and Nelson 1995).
CYP90A,
CYP85, and DWF4, which are thought to be involved in BR metabolism, branched
from CYP88, which mediates GA biosynthesis. GenBank accession numbers for the
group A cytochrome P450s are M32885 (avocado; CYP71A1), P48421 (Arabidopsis;
CYP83), P48418 (petunia; CYP75A1), and X71658 (eggplant; CYP76A1). The
DISTANCE utility in the Genetics Computer Group software package was employed
to calculate the relationships.
Figure 5 depicts a comparison of wild-type and dwf4 hypocotyl growth rates.
Circles indicate wild-type and square indicate dwf4. Each data point
represents the
average of 10 seedlings.
Figure 6 depicts responses to cell elongation signals. BL measurements were
performed with dwf4-3 and the corresponding wild-type control, Enkheim. Open
bars
indicate the wild type. Filled bars indicate dwf4. Lines above the bars
represent one
standard deviation. On the horizontal axis, "light" refers to light-grown
controls;
"dark" refers to dark-grown controls; "hy2" refers to DWF4 and dwf4 plants in
a hy2
background; "GA" refers to plants grown in 10-5 M GA; "2,4-D" refers to plants
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grown in 10-8 M 2,4-D; "-BR" refers to liquid-grown controls; and "+BR" refers
to
liquid-grown controls with 10-a M BL.
Figure 7 depicts pedicel elongation of dwf4 mature plants in response to
exogenous application of BR. Measurements were performed with the BR-fed
plants.
dwf4-1 plants were more sensitive to intermediates belonging to the late C-6
oxidation
pathway (10-6 M 6-deoxoCT and 10-6 M 6-deoxoTE) compared with compounds in
the early C-6 pathway (10'5 M CT and 10'5 M TE). BL (10-' M) induced almost
the
same amount of elongation with one-tenth the concentration of its precursors.
Rescue
by 22-OHCR (10-5 M), which is structurally similar to the presumed precursor
CR,
except for a 22a-hydroxyl functional group, shows that the only defect in dwf4
is the
C-22 hydroxylation reaction. Complementing intermediates and BL induced
dramatic
elongation in the elongating zone of the inflorescence and pedicel, but
fertility was
not increased. Data represent the means tSE of 15 to 20 pedicels. "CTRL"
refers to
control; "WT" refers to wild type.
Figure 8 depicts the increase in inflorescence growth of three transformants
which overexpress dwf4 as compared to wild type (Ws-2). The length of
inflorescences of DWF4 overexpression lines increased more than 20% compared
to
that of wild type. The length of the plant was measured at maturity. Each date
point
is a mean value of more than 9 plants, except AOD4-60 which represents 2
plants.
Figure 9 depicts the increase in seed production of three transformants which
overexpress dwf4 as compared to wild type (Ws-2). Seeds were harvested from
individual plants of each genotype (n>5). Seeds from each plant were weighed
and a
mean value calculated. The Figure shows percent increase over wild type.
Figures 10(A)-10(G) depict the nucleotide sequence of wild-type dwf4 (SEQ
ID NO:1, see, also, GenBank Accession Number AF044216). The dwf4
polynucleotide includes a coding region between nucleotides 3203 and 6110,
inclusive. The coding region includes the following eight exons: nucleotides
3203 to
3423, inclusive; nucleotides 3504 to 3828, inclusive; nucleotides 3914 to
4066,
inclusive; nucleotides 4165 to 4479, inclusive; nucleotides 4632 to 4724,
inclusive;
nucleotides 4816 to 4894, inclusive; nucleotides 5001 to 5110, inclusive and
nucleotides 5865 to 6110, inclusive. The exons are indicated by a bar beneath
the
nucleotide sequence. A 5' control region (e.g., promoter) extends from
nucleotides 1
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to 3202. A 3' untranslated region (UTR), corresponds to the region extending
from
nucleotide to 6011 to approximately nucleotide 6468 of Figure 10 (SEQ ID NO:1
)
and a TATA signal extending approximately from nucleotides 3060 to 3125. As
described in the Examples, mutant alleles of dwf4 have also been
characterized. For
example, dwf4-1 contains an approximately 20 kb insert between nucleotides
5202
and 5203. dwf4-2 has a 9 base pair deletion corresponding to amino acids 324-
326.
In mutant allele dwf4-3, the guanine (G) residue at position 4332 is replaced
with an
adenine (A) residue to create a premature stop codon and truncate the DWF4
protein
at amino acid 289.
Figure 11 depicts the amino acid sequence of the DWF4 polypeptide
(GenBank Accession Number AAC05093, SEQ ID N0:2). The polypeptide is 513
amino acids in length.
Figure 12 depicts seedling phenotypes of twelve-day-old dwf4-l, wild type,
epi-BL-treated wild type, and ADD4 lines grown in the light and dark,
particularly
quantification of hypocotyl and root growth. The average lengths of 16
seedlings are
displayed with the standard deviation. Increased BR concentration supplied
exogenously or endogenously resulted in both elongated hypocotyls and
shortened
roots.
DETAILED DESCRIPTION
The novel dwf4 polynucleotides and DWF4 polypeptides described herein are
important molecules in regulating cell growth and sterol synthesis. The
present
inventors have shown that dwf4 encodes a cytochrome P450 monooxygenase having
43% sequence identity to the protein termed Constitutive Phoromorphogenesis
and
Dwfarism (CPD). As shown in Figure 1, both CPD and DWF4 polypeptides appear
to regulate biosynthesis of brassinosteriods, for example brassinolide (BL).
However,
unlike previously characterized proteins (e.g, CPD), DWF4 appears to act as a
"gatekeeper" in these biosynthetic pathways in that its substrates (e.g., 6-
Oxo
campestanol and 6a-Hydroxy campestanol) are approximately 500 times more
prevalent than the downstream molecules. Thus, the present invention
represents an
important discovery in understanding and regulating cell growth.
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Before describing the present invention in detail, it is to be understood that
this invention is not limited to particularly exemplified molecules or process
parameters as such may, of course, vary. It is also to be understood that the
terminology used herein is for the purpose of describing particular
embodiments of
the invention only, and is not intended to be limiting. In addition, the
practice of the
present invention will employ, unless otherwise indicated, conventional
methods of
plant biology, virology, microbiology, molecular biology, recombinant DNA
techniques and immunology all of which are within the ordinary skill of the
art. Such
techniques are explained fully in the literature. See, e.g., Evans, et al.,
Handbook of
Plant Cell Culture (1983, Macmillan Publishing Co.); Binding, Regeneration of
Plants, Plant Protoplasts (1985, CRC Press); Sambrook, et al., Molecular
Cloning: A
Laboratory Manual (2nd Edition, 1989); DNA Cloning: A Practical Approach, vol.
I
& II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., 1984); A
Practical
Guide to Molecular Cloning (1984); and Fundamental Virology, 2nd Edition, vol.
I &
II (B.N. Fields and D.M. Knipe, eds.).
It must be noted that, as used in this specification and the appended claims,
the
singular forms "a", "an" and "the" include plural referents unless the content
clearly
dictates otherwise. Thus, for example, reference to "a polypeptide" includes a
mixture
of two or more polypeptides, and the like.
The following amino acid abbreviations are used throughout the text:
Alanine: Ala (A) Arginine: Arg
(R)
Asparagine: Asn (N) Aspartic acid:
Asp (D)
Cysteine: Cys (C) Glutamine: Gln
(Q)
Glutamic acid: Glu (E) Glycine: Gly (G)
Histidine: His (H) Isoleucine: Ile
(I)
Leucine: Leu (L) Lysine: Lys (K)
Methionine: Met (M) Phenylalanine:
Phe (F)
Proline: Pro (P) Serine: Ser (S)
Threonine: Thr (T) Tryptophan: Trp
(W)
Tyrosine: Tyr (Y) Valine: Val (V)
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Definitions
In describing the present invention, the following terms will be employed, and
are intended to be defined as indicated below.
The terms "nucleic acid molecule" and "polynucleotide" are used
interchangeably and refer to a polymeric form of nucleotides of any length,
either
deoxyribonucleotides or ribonucleotides, or analogs thereof. This term refers
only to
the primary structure of the molecule and thus includes double- and single-
stranded
DNA and RNA. It also includes known types of modifications, for example,
labels
which are known in the art, methylation, "caps", substitution of one or more
of the
naturally occurring nucleotides with an analog, internucleotide modifications
such as,
for example, those with uncharged linkages (e.g., methyl phosphonates,
phosphotriesters, phosphoamidates, carbamates, etc.) and with charged linkages
(e.g.,
phosphorothioates, phosphorodithioates, etc.), those containing pendant
moieties,
such as, for example proteins (including e.g., nucleases, toxins, antibodies,
signal
peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine,
psoralen, etc.),
those containing chelates (e.g., metals, radioactive metals, boron, oxidative
metals,
etc.), those containing alkylators, those with modified linkages (e.g., alpha
anomeric
nucleic acids, etc.), as well as unmodified forms of the polynucleotide.
Polynucleotides may have any three-dimensional structure, and may perform any
function, known or unknown. Nonlimiting examples of polynucleotides include a
gene, a gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA,
ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched
polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA
of
any sequence, nucleic acid probes, and primers.
A polynucleotide is typically composed of a specific sequence of four
nucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine (T)
(uracil (U)
for thymine (T) when the polynucleotide is RNA). Thus, the term polynucleotide
sequence is the alphabetical representation of a polynucleotide molecule. This
alphabetical representation can be input into databases in a computer having a
central
processing unit and used for bioinformatics applications such as functional
genomics
and homology searching.
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Techniques for determining nucleic acid and amino acid "sequence identity"
are known in the art. Typically, such techniques include determining the
nucleotide
sequence of the mRNA for a gene and/or determining the amino acid sequence
encoded thereby, and comparing these sequences to a second nucleotide or amino
acid
sequence. In general, "identity" refers to an exact nucleotide-to-nucleotide
or amino
acid-to-amino acid correspondence of two polynucleotides or polypeptide
sequences,
respectively. Two or more sequences (polynucleotide or amino acid) can be
compared by determining their "percent identity." The percent identity of two
sequences, whether nucleic acid or amino acid sequences, is the number of
exact
matches between two aligned sequences divided by the length of the shorter
sequences and multiplied by 100. An approximate alignment for nucleic acid
sequences is provided by the local homology algorithm of Smith and Waterman,
Advances in Applied Mathematics 2:482-489 (1981). This algorithm can be
applied
to amino acid sequences by using the scoring matrix developed by Dayhoff,
Atlas of
Protein Sequences and Structure, M.O. Dayhoff ed., 5 suppl. 3:353-358,
National
Biomedical Research Foundation, Washington, D.C., USA, and normalized by
Gribskov, Nucl. Acids Res. 14(6):6745-6763 (1986). An exemplary implementation
of this algorithm to determine percent identity of a sequence is provided by
the
Genetics Computer Group (Madison, WI) in the "BestFit" utility application.
The
default parameters for this method are described in the Wisconsin Sequence
Analysis
Package Program Manual, Version 8 (1995) (available from Genetics Computer
Group, Madison, WI). A preferred method of establishing percent identity in
the
context of the present invention is to use the MPSRCH package of programs
copyrighted by the University of Edinburgh, developed by John F. Collins and
Shane
S. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View, CA). From
this
suite of packages the Smith-Waterman algorithm can be employed where default
parameters are used for the scoring table (for example, gap open penalty of
12, gap
extension penalty of one, and a gap of six). From the data generated the
"Match"
value reflects "sequence identity." Other suitable programs for calculating
the percent
identity or similarity between sequences are generally known in the art, for
example,
another alignment program is BLAST, used with default parameters. For example,
BLASTN and BLASTP can be used using the following default parameters: genetic
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code = standard; filter = none; strand = both; cutoff = 60; expect = 10;
Matrix =
BLOSUM62; Descriptions = SO sequences; sort by = HIGH SCORE; Databases =
non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations +
Swiss protein + Spupdate + PIR. Details of these programs can be found at the
following Internet address: http://www.ncbi.nlm.gov/cgi-bin/BLAST.
Alternatively, the degree of sequence similarity between polynucleotides can
be determined by hybridization of polynucleotides under conditions that form
stable
duplexes between homologous regions, followed by digestion with single-
stranded-
specific nuclease(s), and size determination of the digested fragments. Two
DNA, or
two polypeptide sequences are "substantially homologous" to each other when
the
sequences exhibit at least about 43%-60%, preferably 60-70%, more preferably
70%
85%, more preferably at least about 85%-90%, more preferably at least about
90%
95%, and most preferably at least about 95%-98% sequence identity over a
defined
length of the molecules, or any percentage between the above-specified ranges,
as
determined using the methods above. As used herein, substantially homologous
also
refers to sequences showing complete identity to the specified DNA or
polypeptide
sequence. DNA sequences that are substantially homologous can be identified in
a
Southern hybridization experiment under, for example, stringent conditions, as
defined for that particular system. Defining appropriate hybridization
conditions is
within the skill of the art. See, e.g., Sambrook et al., supra; DNA Cloning,
supra;
Nucleic Acid Hybridization, supra.
The degree of sequence identity between two nucleic acid molecules affects
the efficiency and strength of hybridization events between such molecules. A
partially identical nucleic acid sequence will at least partially inhibit a
completely
identical sequence from hybridizing to a target molecule. Inhibition of
hybridization
of the completely identical sequence can be assessed using hybridization
assays that
are well known in the art (e.g., Southern blot, Northern blot, solution
hybridization, or
the like, see Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second
Edition, (1989) Cold Spring Harbor, N.Y.). Such assays can be conducted using
varying degrees of selectivity, for example, using conditions varying from low
to high
stringency. If conditions of low stringency are employed, the absence of non-
specific
binding can be assessed using a secondary probe that lacks even a partial
degree of
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sequence identity (for example, a probe having less than about 30% sequence
identity
with the target molecule), such that, in the absence of non-specific binding
events, the
secondary probe will not hybridize to the target.
When utilizing a hybridization-based detection system, a nucleic acid probe is
S chosen that is complementary to a target nucleic acid sequence, and then by
selection
of appropriate conditions the probe and the target sequence "selectively
hybridize," or
bind, to each other to form a hybrid molecule. A nucleic acid molecule that is
capable
of hybridizing selectively to a target sequence under "moderately stringent"
typically
hybridizes under conditions that allow detection of a target nucleic acid
sequence of at
least about 10-14 nucleotides in length having at least approximately 70%
sequence
identity with the sequence of the selected nucleic acid probe. Stringent
hybridization
conditions typically allow detection of target nucleic acid sequences of at
least about
10-14 nucleotides in length having a sequence identity of greater than about
90-95%
with the sequence of the selected nucleic acid probe. Hybridization conditions
useful
for probe/target hybridization where the probe and target have a specific
degree of
sequence identity, can be determined as is known in the art (see, for example,
Nucleic
Acid Hybridization: A Practical Approach, editors B.D. Hames and S.J. Higgins,
(1985) Oxford; Washington, DC; IRL Press).
With respect to stringency conditions for hybridization, it is well known in
the
art that numerous equivalent conditions can be employed to establish a
particular
stringency by varying, for example, the following factors: the length and
nature of
probe and target sequences, base composition of the various sequences,
concentrations of salts and other hybridization solution components, the
presence or
absence of blocking agents in the hybridization solutions (e.g., formamide,
dextran
sulfate, and polyethylene glycol), hybridization reaction temperature and time
parameters, as well as, varying wash conditions. The selection of a particular
set of
hybridization conditions is selected following standard methods in the art
(see, for
example, Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second
Edition, (1989) Cold Spring Harbor, N.Y.).
A "gene" as used in the context of the present invention is a sequence of
nucleotides in a genetic nucleic acid (chromosome, plasmid, etc.) with which a
genetic function is associated. A gene is a hereditary unit, for example of an
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organism, comprising a polynucleotide sequence that occupies a specific
physical
location (a "gene locus" or "genetic locus") within the genome of an organism.
A
gene can encode an expressed product, such as a polypeptide or a
polynucleotide (e.g.,
tRNA). Alternatively, a gene may define a genomic location for a particular
event/function, such as the binding of proteins and/or nucleic acids, wherein
the gene
does not encode an expressed product. Typically, a gene includes coding
sequences,
such as, polypeptide encoding sequences, and non-coding sequences, such as,
promoter sequences, polyadenlyation sequences, transcriptional regulatory
sequences
(e.g., enhancer sequences). Many eucaryotic genes have "exons" (coding
sequences)
interrupted by "introns" (non-coding sequences). In certain cases, a gene may
share
sequences with another genes) (e.g., overlapping genes).
A "coding sequence" or a sequence which "encodes" a selected polypeptide, is
a nucleic acid molecule which is transcribed (in the case of DNA) and
translated (in
the case of mRNA) into a polypeptide, for example, in vivo when placed under
the
control of appropriate regulatory sequences (or "control elements"). The
boundaries
of the coding sequence are typically determined by a start codon at the S'
(amino)
terminus and a translation stop codon at the 3' (carboxy) terminus. A coding
sequence
can include, but is not limited to, cDNA from viral, procaryotic or eucaryotic
mRNA,
genomic DNA sequences from viral or procaryotic DNA, and even synthetic DNA
sequences. A transcription termination sequence may be located 3' to the
coding
sequence. Other "control elements" may also be associated with a coding
sequence.
A DNA sequence encoding a polypeptide can be optimized for expression in a
selected cell by using the codons preferred by the selected cell to represent
the DNA
copy of the desired polypeptide coding sequence. "Encoded by" refers to a
nucleic
acid sequence which codes for a polypeptide sequence, wherein the polypeptide
sequence or a portion thereof contains an amino acid sequence of at least 3 to
5 amino
acids, more preferably at least 8 to 10 amino acids, and even more preferably
at least
15 to 20 amino acids from a polypeptide encoded by the nucleic acid sequence.
Also
encompassed are polypeptide sequences which are immunologically identifiable
with
a polypeptide encoded by the sequence.
Typical "control elements", include, but are not limited to, transcription
promoters, transcription enhancer elements, transcription termination signals,
CA 02362603 2001-08-10
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polyadenylation sequences (located 3' to the translation stop codon),
sequences for
optimization of initiation of translation (located 5' to the coding sequence),
translation
enhancing sequences, and translation termination sequences. Transcription
promoters
can include inducible promoters (where expression of a polynucleotide sequence
operably linked to the promoter is induced by an analyte, cofactor, regulatory
protein,
etc.), tissue-specific promoters (where expression of a polynucleotide
sequence
operably linked to the promoter is induced only in selected tissue),
repressible
promoters (where expression of a polynucleotide sequence operably linked to
the
promoter is induced by an analyte, cofactor, regulatory protein, etc.), and
constitutive
promoters.
A control element, such as a promoter, "directs the transcription" of a coding
sequence in a cell when RNA polymerase will bind the promoter and transcribe
the
coding sequence into mRNA, which is then translated into the polypeptide
encoded
by the coding sequence.
"Expression enhancing sequences" typically refer to control elements that
improve transcription or translation of a polynucleotide relative to the
expression
level in the absence of such control elements (for example, promoters,
promoter
enhancers, enhancer elements, and translational enhancers (e.g., Shine and
Delagarno
sequences).
"Operably linked" refers to a juxtaposition wherein the components so
described are in a relationship permitting them to function in their intended
manner.
A control sequence "operably linked" to a coding sequence is ligated in such a
way
that expression of the coding sequence is achieved under conditions compatible
with
the control sequences. The control elements need not be contiguous with the
coding
sequence, so long as they function to direct the expression thereof. Thus, for
example, intervening untranslated yet transcribed sequences can be present
between a
promoter and the coding sequence and the promoter can still be considered
"operably
linked" to the coding sequence.
A "heterologous sequence" as used herein typically refers to a nucleic acid
sequence that is not normally found in the cell or organism of interest. For
example, a
DNA sequence encoding a polypeptide can be obtained from a plant cell and
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introduced into a bacterial cell. In this case the plant DNA sequence is
"heterologous" to the native DNA of the bacterial cell.
The "native sequence" or "wild-type sequence" o#~ a gene is the polynucleotide
sequence that comprises the genetic locus corresponding to the gene, e.g., all
regulatory and open-reading frame coding sequences required for expression of
a
completely functional gene product as they are present in the wild-type genome
of an
organism. The native sequence of a gene can include, for example,
transcriptional
promoter sequences, translation enhancing sequences, introns, exons, and poly-
A
processing signal sites. It is noted that in the general population, wild-type
genes may
include multiple prevalent versions that contain alterations in sequence
relative to
each other and yet do not cause a discernible pathological effect. These
variations are
designated "polymorphisms" or "allelic variations."
"Recombinant" as used herein to describe a nucleic acid molecule means a
polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin which, by
virtue
of its origin or manipulation: (1) is not associated with all or a portion of
the
polynucleotide with which it is associated in nature; and/or (2) is linked to
a
polynucleotide other than that to which it is linked in nature. The term
"recombinant"
as used with respect to a protein or polypeptide means a polypeptide produced
by
expression of a recombinant polynucleotide.
By "vector" is meant any genetic element, such as a plasmid, phage,
transposon, cosmid, chromosome, virus etc., which is capable of transferring
gene
sequences to target cells. Generally, a vector is capable of replication when
associated with the proper control elements. Thus, the term includes cloning
and
expression vehicles, as well as viral vectors and integrating vectors.
As used herein, the term "expression cassette" refers to a molecule comprising
at least one coding sequence operably linked to a control sequence which
includes all
nucleotide sequences required for the transcription of cloned copies of the
coding
sequence and the translation of the mRNAs in an appropriate host cell. Such
expression cassettes can be used to express eukaryotic genes in a variety of
hosts such
as bacteria, blue-green algae, plant cells, yeast cells, insect cells and
animal cells.
Under the invention, expression cassettes can include, but are not limited to,
cloning
vectors, specifically designed plasmids, viruses or virus particles. The
cassettes may
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further include an origin of replication for autonomous replication in host
cells,
selectable markers, various restriction sites, a potential for high copy
number and
strong promoters.
A cell has been "transformed" by an exogenous polynucleotide when the
polynucleotide has been introduced inside the cell. The exogenous
polynucleotide
may or may not be integrated (covalently linked) into chromosomal DNA making
up
the genome of the cell. In prokaryotes and yeasts, for example, the exogenous
DNA
may be maintained on an episomal element, such as a plasmid. With respect to
eucaryotic cells, a stably transformed cell is one in which the exogenous DNA
has
become integrated into the chromosome so that it is inherited by daughter
cells
through chromosome replication. This stability is demonstrated by the ability
of the
eucaryotic cell to establish cell lines or clones comprised of a population of
daughter
cells containing the exogenous DNA.
"Recombinant host cells " "host cells " "cells " "cell lines " "cell cultures
" and
> > > > >
other such terms denoting procaryotic microorganisms or eucaryotic cell lines
cultured as unicellular entities, are used interchangeably, and refer to cells
which can
be, or have been, used as recipients for recombinant vectors or other transfer
DNA,
and include the progeny of the original cell which has been transfected. It is
understood that the progeny of a single parental cell may not necessarily be
completely identical in morphology or in genomic or total DNA complement to
the
original parent, due to accidental or deliberate mutation. Progeny of the
parental cell
which are sufficiently similar to the parent to be characterized by the
relevant
properly, such as the presence of a nucleotide sequence encoding a desired
peptide,
are included in the progeny intended by this definition, and are covered by
the above
terms.
The term "dwf4 polynucleotide" refers to a polynucleotide derived from the
dwf4 gene. The gene encodes the protein referred to herein as DWF4. DWF4 is a
cytochrome P450 cytochrome P450 that mediates multiple 22a-hydroxylation steps
in
brassinosteroid biosynthesis (see, Figure 1). The dwf4 polynucleotide sequence
and
corresponding amino acid sequence are shown in Figures 10 and 11 (SEQ ID NO:1,
SEQ 117 N0:2 and GenBank accession No. AF044216). As shown in Figure 10, the
dwf4 coding sequence spans the region from nucleotide positions 3203 to 6110
and
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the upstream 5' UTR, including the promoter region, spans nucleotide positions
1 to
3202. A functional 1.1 kb control element is also described in the Examples. A
3'
UTR spans nucleotide positions 6111 to approximately 6468 of SEQ ID NO:I . The
term as used herein encompasses a polynucleotide including a native sequence
depicted in Figure 10, as well as modifications and fragments thereof.
The term encompasses alterations to the polynucleotide sequence, so long as
the alteration results in a plant displaying one or more dwf4 phenotypic
traits
(described below) when the polynucleotide is expressed in a plant. Such
modifications typically include deletions, additions and substitutions, to the
native
dwf4 sequence, so long as the mutation results in a plant displaying a dwf4
phenotype
as defined below. These modifications may be deliberate, as through site-
directed
mutagenesis, or may be accidental, such as through mutations of plants which
express
the dwf4 polynucleotide or errors due to PCR amplification. The term
encompasses
expressed allelic variants of the wild-type dwf4 sequence which may occur by
normal
genetic variation or are produced by genetic engineering methods and which
result in
a detectable change in the wild-type dwf4 phenotype.
The term "dwf4 phenotype" as used herein refers to any microscopic or
macroscopic change in structure or morphology of a plant, such as a transgenic
plant,
as well as biochemical differences, which are characteristic of a dwf4 plant,
compared
to a progenitor, wild-type plant cultivated under the same conditions.
Generally,
morphological differences include multiple short stems, short rounded leaves,
loss of
fertility due to reduced stamen length, and delayed development. Dark-grown
dwf4
seedlings possess short hypocotyls, open cotyledons, and developing leaves.
The
height of such plants will typically be 75% or less of the wild-type plant,
more
typically 50% or less of the wild-type plant, and even more typically 25% or
less of
the wild-type plant, or any integer in between. Additional phenotypic
morphological
attributes of the dwf4 mutant are summarized in Table 1 of the examples.
Biochemically, dwf4 hypocotyls are converted to wild-type length with the
application of BL.
A "polypeptide" is used in it broadest sense to refer to a compound of two or
more subunit amino acids, amino acid analogs, or other peptidomimetics. The
subunits may be linked by peptide bonds or by other bonds, for example ester,
ether,
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etc. As used herein, the term "amino acid" refers to either natural and/or
unnatural or
synthetic amino acids, including glycine and both the D or L optical isomers,
and
amino acid analogs and peptidomimetics. A peptide of three or more amino acids
is
commonly called an oligopeptide if the peptide chain is short. If the peptide
chain is
long, the peptide is typically called a polypeptide or a protein. Full-length
proteins,
analogs, mutants and fragments thereof are encompassed by the definition. The
terms
also include postexpression modifications of the polypeptide, for example,
glycosylation, acetylation, phosphorylation and the like. Furthermore, as
ionizable
amino and carboxyl groups are present in the molecule, a particular
polypeptide may
be obtained as an acidic or basic salt, or in neutral form. A polypeptide may
be
obtained directly from the source organism, or may be recombinantly or
synthetically
produced (see further below).
A "DWF4" polypeptide is a polypeptide as defined above, which is derived
from a 22a-hydroxylase that functions in the brassinolide (BL) biosynthetic
pathway
(see, Figure 1). The native sequence of full-length DWF4 is shown in Figure 11
(SEQ ID N0:2). However, the term encompasses mutants and fragments of the
native
sequence so long as the protein functions for its intended purpose.
The term "DWF4 analog" refers to derivatives of DWF4, or fragments of such
derivatives, that retain desired function, e.g., as measured in assays as
described
further below. In general, the term "analog" refers to compounds having a
native
polypeptide sequence and structure with one or more amino acid additions,
substitutions (generally conservative in nature) and/or deletions, relative to
the native
molecule, so long as the modifications do not destroy desired activity.
Preferably,
the analog has at least the same activity as the native molecule. Methods for
making
polypeptide analogs are known in the art and are described further below.
Particularly preferred analogs include substitutions that are conservative in
nature, i.e., those substitutions that take place within a family of amino
acids that are
related in their side chains. Specifically, amino acids are generally divided
into four
families: (1) acidic -- aspartate and glutamate; (2) basic -- lysine,
arginine, histidine;
(3) non-polar -- alanine, valine, leucine, isoleucine, proline, phenylalanine,
methionine, tryptophan; and (4) uncharged polar -- glycine, asparagine,
glutamine,
cysteine, serine threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine
are
CA 02362603 2001-08-10
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sometimes classified as aromatic amino acids. For example, it is reasonably
predictable that an isolated replacement of leucine with isoleucine or valine,
an
aspartate with a glutamate, a threonine with a serine, or a similar
conservative
replacement of an amino acid with a structurally related amino acid, will not
have a
major effect on the biological activity. It is to be understood that the terms
include
the various sequence polymorphisms that exist, wherein amino acid
substitutions in
the protein sequence do not affect the essential functions of the protein.
By "purified" and "isolated" is meant, when referring to a polypeptide or
polynucleotide, that the molecule is separate and discrete from the whole
organism
with which the molecule is found in nature; or devoid, in whole or part, of
sequences
normally associated with it in nature; or a sequence, as it exists in nature,
but having
heterologous sequences (as defined below) in association therewith. It is to
be
understood that the term "isolated" with reference to a polynucleotide intends
that the
polynucleotide is separate and discrete from the chromosome from which the
polynucleotide may derive. The term "purified" as used herein preferably means
at
least 75% by weight, more preferably at least 85% by weight, more preferably
still at
least 95% by weight, and most preferably at least 98% by weight, of biological
macromolecules of the same type are present. An "isolated polynucleotide which
encodes a particular polypeptide" refers to a nucleic acid molecule which is
substantially free of other nucleic acid molecules that do not encode the
subject
polypeptide; however, the molecule may include some additional bases or
moieties
which do not deleteriously affect the basic characteristics of the
composition.
By "fragment" is intended a polypeptide or polynucleotide consisting of only a
part of the intact sequence and structure of the reference polypeptide or
polynucleotide, respectively. The fragment can include a 3' or C-terminal
deletion or
a 5' or N-terminal deletion, or even an internal deletion, of the native
molecule. A
polynucleotide fragment of a dwf4 sequence will generally include at least
about 15
contiguous bases of the molecule in question, more preferably 18-25 contiguous
bases, even more preferably 30-50 or more contiguous bases of the dwf4
molecule, or
any integer between 15 bases and the full-length sequence of the molecule.
Fragments which provide at least one dwf4 phenotype as defined above are
useful in
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the production of transgenic plants. Fragments are also useful as
oligonucleotide
probes, to find additional dwf4 sequences.
Similarly, a polypeptide fragment of a DWF4 molecule will generally include
at least about 5-10 contiguous amino acid residues of the full-length
molecule,
preferably at least about 1 S-25 contiguous amino acid residues of the full-
length
molecule, and most preferably at least about 20-50 or more contiguous amino
acid
residues of the full-length DWF4 molecule, or any integer between 10 amino
acids
and the full-length sequence of the molecule. Such fragments are useful for
the
production of antibodies and the like.
By "transgenic plant" is meant a plant into which one or more exogenous
polynucleotides have been introduced. Examples of means by which this can be
accomplished are described below, and include Agrobacterium-mediated
transformation, biolistic methods, electroporation, and the like. In the
context of the
present invention, the transgenic plant contains a polynucleotide which is not
normally present in the corresponding wild-type plant and which confers at
least one
dwf4 phenotypic trait to the plant. The transgenic plant therefore exhibits
altered
structure, morphology or biochemistry as compared with a progenitor plant
which
does not contain the transgene, when the transgenic plant and the progenitor
plant are
cultivated under similar or equivalent growth conditions. Such a plant
containing the
exogenous polynucleotide is referred to here as an R, generation transgenic
plant.
Transgenic plants may also arise from sexual cross or by selfing of transgenic
plants
into which exogenous polynucleotides have been introduced. Such a plant
containing
the exogenous nucleic acid is also referred to here as an R, generation
transgenic
plant. Transgenic plants which arise from a sexual cross with another parent
line or by
selfing are "descendants or the progeny" of a R, plant and are generally
called Fn
plants or Sn plants, respectively, n meaning the number of generations.
General Overview
In this report, we present morphological, biochemical, and molecular analysis
of a novel gene, dwf4, isolated from Arabidopsis. Morphologically, dwf4 plants
display a dramatic reduction in the length of many different organs examined,
and this
size reduction is attributable to a defect in cell elongation. Biochemically,
dwf4
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hypocotyls were converted completely to wild-type length with the application
of BL,
suggesting a deficiency in BRs. In agreement with this, BR intermediate
feeding
analysis, indicated that dwf4 encodes a cytochrome P450 that mediates multiple
22a-
hydroxylation steps in brassinosteriod biosynthesis. Sequencing of the dwf4
locus
and analysis of the protein product are described.
The molecules of the present invention are therefore useful in the production
of transgenic plants which display at least one dwf4 phenotype, so that the
resulting
plants have altered structure or morphology. The present invention
particularly
provides for altered structure or morphology such as reduced cell length,
extended
flowering periods, increased size of leaves or fruit, increased branching,
increased
seed production and altered sterol composition relative wild-type plants. The
DWF4
polypeptides can be expressed to engineer a plant with desirable properties.
The
engineering is accomplished by transforming plants with nucleic acid
constructs
described herein which may also comprise promoters and secretion signal
peptides.
The transformed plants or their progenies are screened for plants that express
the
desired polypeptide.
Engineered plants exhibiting the desired altered structure or morphology can
be used in plant breeding or directly in agricultural production or industrial
applications. Plants having the altered polypeptide can be crossed with other
altered
plants engineered with alterations in other growth modulation enzymes,
proteins or
polypeptides to produce lines with even further enhanced altered structural
morphology characteristics compared to the parents or progenitor plants.
Isolation of Nucleic Acid Sequences from Plants
The isolation of dwf4 sequences from the polynucleotides of the invention
may be accomplished by a number of techniques. For instance, oligonucleotide
probes based on the sequences disclosed here can be used to identify the
desired gene
in a cDNA or genomic DNA library from a desired plant species. To construct
genomic libraries, large segments of genomic DNA are generated by random
fragmentation, e.g. using restriction endonucleases, and are ligated with
vector DNA
to form concatemers that can be packaged into the appropriate vector. To
prepare a
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library of tissue-specific cDNAs, mRNA is isolated from tissues and a cDNA
library
which contains the gene transcripts is prepared from the mRNA.
The cDNA or genomic library can then be screened using a probe based upon
the sequence of a cloned gene such as the polynucleotides disclosed here.
Probes may
be used to hybridize with genomic DNA or cDNA sequences to isolate homologous
genes in the same or different plant species. Alternatively, the nucleic acids
of
interest can be amplified from nucleic acid samples using amplification
techniques.
For instance, polymerase chain reaction (PCR) technology to amplify the
sequences
of the genes directly from mRNA, from cDNA, from genomic libraries or cDNA
libraries. PCR® and other in vitro amplification methods may also be
useful, for
example, to clone nucleic acid sequences that code for proteins to be
expressed, to
make nucleic acids to use as probes for detecting the presence of the desired
mRNA in
samples, for nucleic acid sequencing, or for other purposes.
Appropriate primers and probes for identifying dwf4-specific genes from plant
tissues are generated from comparisons of the sequences provided herein. For a
general overview of PCR see Innis et al. eds, PCR Protocols: A Guide to
Methods and
Applications, Academic Press, San Diego (1990). Appropriate primers for this
invention include, for instance, those primers described in the Examples and
Sequence
Listings, as well as other primers derived from the dwf4 sequences disclosed
herein.
Suitable amplifications conditions may be readily determined by one of skill
in the art
in view of the teachings herein, for example, including reaction components
and
amplification conditions as follows: 10 mM Tris-HCI, pH 8.3, 50 mM potassium
chloride, 1.5 mM magnesium chloride, 0.001 % gelatin, 200 ~M dATP, 200 ~M
dCTP, 200 qM dGTP, 200 pM dTTP, 0.4 pM primers, and 100 units per mL Taq
polymerase; 96°C for 3 min., 30 cycles of 96°C for 45 seconds,
50°C for 60 seconds,
72 ° C for 60 seconds, followed by 72 ° C for 5 min.
Polynucleotides may also be synthesized by well-known techniques as
described in the technical literature. See, e.g., Carruthers, et al. (1982)
Cold Spring
Harbor Symp. Quant. Biol. 47:411-418, and Adams, et al. (1983) J. Am. Chem.
Soc.
105:661. Double stranded DNA fragments may then be obtained either by
synthesizing the complementary strand and annealing the strands together under
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appropriate conditions, or by adding the complementary strand using DNA
polymerase with an appropriate primer sequence.
The polynucleotides of the present invention may also be used to isolate or
create other mutant cell gene alleles. Mutagenesis consists primarily of site-
directed
mutagenesis followed by phenotypic testing of the altered gene product. Some
of the
more commonly employed site-directed mutagenesis protocols take advantage of
vectors that can provide single stranded as well as double stranded DNA, as
needed.
Generally, the mutagenesis protocol with such vectors is as follows. A
mutagenic
primer, i.e., a primer complementary to the sequence to be changed, but
consisting of
one or a small number of altered, added, or deleted bases, is synthesized. The
primer
is extended in vitro by a DNA polymerase and, after some additional
manipulations,
the now double-stranded DNA is transfected into bacterial cells. Next, by a
variety of
methods, the desired mutated DNA is identified, and the desired protein is
purified
from clones containing the mutated sequence. For longer sequences, additional
1 S cloning steps are often required because long inserts (longer than 2
kilobases) are
unstable in those vectors. Protocols are known to one skilled in the art and
kits for
site-directed mutagenesis are widely available from biotechnology supply
companies,
for example from Amersham Life Science, Inc. (Arlington Heights, Ill.) and
Stratagene Cloning Systems (La Jolla, Cali~).
Control elements
Regulatory regions can be isolated from the dwf4 gene and used in
recombinant constructs for modulating the expression of the dwf4 gene or a
heterologous gene in vitro and/or in vivo. As shown in Figure 10, the coding
region
of the dwf4 gene (designated by the light grey bar) begins at nucleotide
position 1133.
The region of the gene spanning nucleotide positions 990-1132 of Figure 10
includes
the dwf4 promoter. This region may be used in its entirety or fragments of the
region
may be isolated which provide the ability to direct expression of a coding
sequence
linked thereto.
Thus, promoters can be identified by analyzing the 5' sequences of a genomic
clone corresponding to the dwf4-specific genes described here. Sequences
characteristic of promoter sequences can be used to identify the promoter.
Sequences
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controlling eukaryotic gene expression have been extensively studied. For
instance,
promoter sequence elements include the TATA box consensus sequence (TATAAT),
which is usually 20 to 30 base pairs upstream of the transcription start site.
In most
instances the TATA box is required for accurate transcription initiation. In
plants,
further upstream from the TATA box, at positions -80 to -100, there is
typically a
promoter element with a series of adenines surrounding the trinucleotide G (or
T) N
G. (See, J. Messing et al., in Genetic Engineering in Plants, pp. 221-227
(Kosage,
Meredith and Hollaender, eds. (1983)). Methods for identifying and
characterizing
promoter regions in plant genomic DNA are described, for example, in Jordano
et al.
(1989) Plant Cell 1:855-866; Bustos et al (1989) Plant Cell 1:839-854; Green
et al.
(1988) EMBO J. 7:4035-4044; Meier et al. (1991) Plant Cell 3:309-316; and
Zhang et
al (1996) Plant Physiology 110:1069-1079).
Additionally, the promoter region may include nucleotide substitutions,
insertions or deletions that do not substantially affect the binding of
relevant DNA
binding proteins and hence the promoter function. It may, at times, be
desirable to
decrease the binding of relevant DNA binding proteins to "silence" or "down-
regulate" a promoter, or conversely to increase the binding of relevant DNA
binding
proteins to "enhance" or "up-regulate" a promoter. In such instances, the
nucleotide
sequence of the promoter region may be modified by, e.g., inserting additional
nucleotides, changing the identity of relevant nucleotides, including use of
chemically-modified bases, or by deleting one or more nucleotides.
Promoter function can be assayed by methods known in the art, preferably by
measuring activity of a reporter gene operatively linked to the sequence being
tested
for promoter function. Examples of reporter genes include those encoding
luciferase,
green fluorescent protein, GUS, neo, cat and bar.
Polynucleotides comprising untranslated (UTR) sequences and intron/exon
junctions are also within the scope of the invention. UTR sequences include
introns
and 5' or 3' untranslated regions ( S' UTRs or 3' UTRs). As shown in Figures 2
and
10, the dwf4 gene sequence includes eight exons and seven introns. These
portions of
the dwf4 gene especially UTRs, can have regulatory functions related to, for
example,
translation rate and mRNA stability. Thus, these portions of the gene can be
isolated
for use as elements of gene constructs for expression of polynucleotides
encoding
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desired polypeptides. The 5' control element region of dwf4 extends from
nucleotides
1 through 3202 of SEQ ID NO:I. Further, as described in Example 11, a 1.1 kb
portion of this region that is directly upstream of the translation initiation
site contains
elements necessary for transcriptional control of dwf4. In contrast, a 280 by
fragment
of the dwf4 control element region that includes the TATA-like region does not
appear to contain all of the necessary transcriptional control elements (see,
Example
11).
Introns of genomic DNA segments may also have regulatory functions.
Sometimes promoter elements, especially transcription enhancer or suppressor
elements, are found within introns. Also, elements related to stability of
heteronuclear RNA and efficiency of transport to the cytoplasm for translation
can be
found in intron elements. Thus, these segments can also find use as elements
of
expression vectors intended for use to transform plants.
The introns, UTR sequences and intron/exon junctions can vary from the
native sequence. Such changes from those sequences preferably will not affect
the
regulatory activity of the UTRs or intron or intron/exon junction sequences on
expression, transcription, or translation. However, in some instances, down-
regulation of such activity may be desired to modulate traits or phenotypic or
in vitro
activity.
Use of Nucleic Acids of the Invention to Inhibit Gene Expression
The isolated sequences prepared as described herein, can be used to prepare
expression cassettes useful in a number of techniques. For example, expression
cassettes of the invention can be used to suppress (underexpress) endogenous
dwf4
gene expression. Inhibiting expression can be useful, for instance, in
suppressing the
phenotype (e.g., dwarf appearance, 22a-hydroxylase activity) exhibited by dwf4
plants. Further, the inhibitory polynucleotides of the present invention can
also be
used in combination with overexpressing constructs described below, for
example,
using suitable tissue-specific promoters linked to polynucleotides described
herein. In
this way, the polynucleotides can be used to promote dwf4 phenotypes (e.g.,
activity)
in selected tissue and, at the same time, inhibit dwf4 phenotypes (e.g.,
activity) in
different tissue(s).
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A number of methods can be used to inhibit gene expression in plants. For
instance, antisense technology can be conveniently used. To accomplish this, a
nucleic acid segment from the desired gene is cloned and operably linked to a
promoter such that the antisense strand of RNA will be transcribed. The
expression
cassette is then transformed into plants and the antisense strand of RNA is
produced.
In plant cells, it has been suggested that antisense RNA inhibits gene
expression by
preventing the accumulation of mRNA which encodes the enzyme of interest, see,
e.g., Sheehy et al (1988) Proc. Nat. Acad. Sci. USA 85:8805-8809, and Hiatt et
al.,
U.S. Patent Number 4,801,340.
The nucleic acid segment to be introduced generally will be substantially
identical to at least a portion of the endogenous gene or genes to be
repressed. The
sequence, however, need not be perfectly identical to inhibit expression. The
vectors
of the present invention can be designed such that the inhibitory effect
applies to other
proteins within a family of genes exhibiting homology or substantial homology
to the
target gene.
For antisense suppression, the introduced sequence also need not be full
length
relative to either the primary transcription product or fully processed mRNA.
Generally, higher homology can be used to compensate for the use of a shorter
sequence. Furthermore, the introduced sequence need not have the same intron
or
exon pattern, and homology of non-coding segments may be equally effective.
Normally, a sequence of between about 30 or 40 nucleotides and about full
length
nucleotides should be used, though a sequence of at least about 100
nucleotides is
preferred, a sequence of at least about 200 nucleotides is more preferred, and
a
sequence of at least about 500 nucleotides is especially preferred. It is to
be
understood that any integer between the above-recited ranges is intended to be
captured herein.
Catalytic RNA molecules or ribozymes can also be used to inhibit expression
of dwf4 genes. It is possible to design ribozymes that specifically pair with
virtually
any target RNA and cleave the phosphodiester backbone at a specific location,
thereby functionally inactivating the target RNA. In carrying out this
cleavage, the
ribozyme is not itself altered, and is thus capable of recycling and cleaving
other
molecules, making it a true enzyme. The inclusion of ribozyme sequences within
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antisense RNAs confers RNA-cleaving activity upon them, thereby increasing the
activity of the constructs.
A number of classes of ribozyrnes have been identified. One class of
ribozymes is derived from a number of small circular RNAs which are capable of
self cleavage and replication in plants. The RNAs replicate either alone
(viroid
RNAs) or with a helper virus (satellite RNAs). Examples include RNAs from
avocado sunblotch viroid and the satellite RNAs from tobacco ringspot virus,
Lucerne
transient streak virus, velvet tobacco mottle virus, solanum nodiflorum mottle
virus
and subterranean clover mottle virus. The design and use of target RNA-
specific
ribozymes is described in Haseloff et al (1988) Nature 334:585-591.
Another method of suppression is sense suppression. Introduction of
expression cassettes in which a nucleic acid is configured in the sense
orientation with
respect to the promoter has been shown to be an effective means by which to
block
the transcription of target genes. For an example of the use of this method to
modulate expression of endogenous genes see, Napoli et al (1990) The Plant
Cell
2:279-289 and U.S. Patent Numbers 5,034,323, 5,231,020, and 5,283,184.
Generally, where inhibition of expression is desired, some transcription of
the
introduced sequence occurs. The effect may occur where the introduced sequence
contains no coding sequence per se, but only intron or untranslated sequences
homologous to sequences present in the primary transcript of the endogenous
sequence. The introduced sequence generally will be substantially identical to
the
endogenous sequence intended to be repressed. This minimal identity will
typically
be greater than about 50%-65%, but a higher identity might exert a more
effective
repression of expression of the endogenous sequences. Substantially greater
identity
of more than about 80% is preferred, though about 95% to absolute identity
would be
most preferred. It is to be understood that any integer between the above-
recited
ranges is intended to be captured herein. As with antisense regulation, the
effect
should apply to any other proteins within a similar family of genes exhibiting
homology or substantial homology.
For sense suppression, the introduced sequence in the expression cassette,
needing less than absolute identity, also need not be full length, relative to
either the
primary transcription product or fully processed mRNA. This may be preferred
to
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avoid concurrent production of some plants which are overexpressers. A higher
identity in a shorter than full length sequence compensates for a longer, less
identical
sequence. Furthermore, the introduced sequence need not have the same intron
or
exon pattern, and identity of non-coding segments will be equally effective.
Normally, a sequence of the size ranges noted above for antisense regulation
is used.
Use of Nucleic Acids of the Invention to Enhance Gene Expression
In addition to inhibiting certain features of a plant, the polynucleotides of
the
invention can be used to increase certain features such as extending
flowering,
producing larger leaves or fruit, producing increased branching and increasing
seed
production. This can be accomplished by the overexpression of dwf4
polynucleotides.
The exogenous dwf4 polynucleotides do not have to code for exact copies of
the endogenous dwf4 proteins. Modified DWF4 protein chains can also be readily
designed utilizing various recombinant DNA techniques well known to those
skilled
in the art and described for instance, in Sambrook et al., supra.
Hydroxylamine can
also be used to introduce single base mutations into the coding region of the
gene
(Sikorski et al (1991) Meth. Enzymol. 194: 302-318). For example, the chains
can
vary from the naturally occurnng sequence at the primary structure level by
amino
acid substitutions, additions, deletions, and the like. These modifications
can be used
in a number of combinations to produce the final modified protein chain.
It will be apparent that the polynucleotides described herein can be used in a
variety of combinations. For example, the polynucleotides can be used to
produce
different phenotypes in the same organism, for instance by using tissue-
specific
promoters to overexpress a dwf4 polynucleotide in certain tissues (e.g., leaf
tissue)
while at the same time using tissue-specific promoters to inhibit expression
of dwf4 in
other tissues. In addition, fusion proteins of the polynucleotides described
herein with
other known polynucleotides (e.g., polynucleotides encoding products involved
in the
BR pathway) can be constructed and employed to obtain desired phenotypes.
Any of the dwf4 polynucleotides described herein can also be used in standard
diagnostic assays, for example, in assays mRNA levels (see, Sambrook et al,
supra);
as hybridization probes, e.g., in combination with appropriate means, such as
a label,
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for detecting hybridization (see, Sambrook et al., supra); as primers, e.g.,
for PCR
(see, Sambrook et al., supra); attached to solid phase supports and the like.
Preparation of Recombinant Vectors
To use isolated sequences in the above techniques, recombinant DNA vectors
suitable for transformation of plant cells are prepared. Techniques for
transforming a
wide variety of higher plant species are well known and described further
below as
well as in the technical and scientific literature. See, for example, Weising
et al (1988)
Ann. Rev. Genet. 22:421-477. A DNA sequence coding for the desired
polypeptide,
for example a cDNA sequence encoding the full length DWF4 protein, will
preferably
be combined with transcriptional and translational initiation regulatory
sequences
which will direct the transcription of the sequence from the gene in the
intended
tissues of the transgenic plant.
Such regulatory elements include but are not limited to the promoters derived
from the genome of plant cells (e.g., heat shock promoters such as soybean
hsp17.5-E
or hspl7.3-B (Gurley et al. (1986) Mol. Cell. Biol. 6:559-565); the promoter
for the
small subunit of RUBISCO (Coruzzi et al. (1984) EMBO J. 3:1671-1680; Broglie
et
al (1984) Science 224:838-843); the promoter for the chlorophyll a/b binding
protein)
or from plant viruses viral promoters such as the 355 RNA and 195 RNA
promoters
of CaMV (Brisson et al. (1984) Nature 310:511-S 14), or the coat protein
promoter of
TMV (Takamatsu et al. (1987) EMBO J. 6:307-311), cytomegalovirus hCMV
immediate early gene, the early or late promoters of SV40 adenovirus, the lac
system,
the trp system, the TAC system, the TRC system, the major operator and
promoter
regions of phage A, the control regions of fd coat protein, the promoter for
3-phosphoglycerate kinase, the promoters of acid phosphatase, heat shock
promoters
(e.g., as described above) and the promoters of the yeast alpha-mating
factors.
In construction of recombinant expression cassettes of the invention, a plant
promoter fragment may be employed which will direct expression of the gene in
all
tissues of a regenerated plant. Such promoters are referred to herein as
"constitutive"
promoters and are active under most environmental conditions and states of
development or cell differentiation. Examples of constitutive promoters
include the
cauliflower mosaic virus (CaMV) 35S transcription initiation region, the T-DNA
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mannopine synthetase promoter (e.g., the 1'- or 2'- promoter derived from T-
DNA of
Agrobacterium tumafaciens), and other transcription initiation regions from
various
plant genes known to those of skill.
Alternatively, the plant promoter may direct expression of the polynucleotide
of the invention in a specific tissue (tissue-specific promoters) or may be
otherwise
under more precise environmental control (inducible promoters). Examples of
tissue-specific promoters under developmental control include promoters that
initiate
transcription only in certain tissues, such as fruit, seeds, or flowers such
as tissue- or
developmental-specific promoter, such as, but not limited to the dwf4
promoter, the
CHS promoter, the PATATIN promoter, etc. The tissue specific E8 promoter from
tomato is particularly useful for directing gene expression so that a desired
gene
product is located in fruits.
Other suitable promoters include those from genes encoding embryonic
storage proteins. Examples of environmental conditions that may affect
transcription
by inducible promoters include anaerobic conditions, elevated temperature, or
the
presence of light. If proper polypeptide expression is desired, a
polyadenylation
region at the 3'-end of the coding region should be included. The
polyadenylation
region can be derived from the natural gene, from a variety of other plant
genes, or
from T-DNA. In addition, the promoter itself can be derived from the dwf4
gene, as
described above.
The vector comprising the sequences (e.g., promoters or coding regions) from
genes of the invention will typically comprise a marker gene which confers a
selectable phenotype on plant cells. For example, the marker may encode
biocide
resistance, particularly antibiotic resistance, such as resistance to
kanamycin, 6418,
bleomycin, hygromycin, or herbicide resistance, such as resistance to
chlorosluforon
or Basta.
Production of Transgenic Plants
DNA constructs of the invention may be introduced into the genome of the
desired plant host by a variety of conventional techniques. For reviews of
such
techniques see, for example, Weissbach & Weissbach Methods for Plant Molecular
Biology (1988, Academic Press, N.Y.) Section VIII, pp. 421-463; and Grierson &
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Corey, Plant Molecular Biology (1988, 2d Ed.), Blackie, London, Ch. 7-9. For
example, the DNA construct may be introduced directly into the genomic DNA of
the
plant cell using techniques such as electroporation and microinjection of
plant cell
protoplasts, or the DNA constructs can be introduced directly to plant tissue
using
biolistic methods, such as DNA particle bombardment (see, e.g., Klein et al
(1987)
Nature 327:70-73). Alternatively, the DNA constructs may be combined with
suitable T-DNA flanking regions and introduced into a conventional
Agrobacterium
tumefaciens host vector. Agrobacterium tumefaciens-mediated transformation
techniques, including disarming and use of binary vectors, are well described
in the
scientific literature. See, for example Horsch et al (1984) Science 233:496-
498, and
Fraley et al (1983) Proc. Nat'l. Acad. Sci. USA 80:4803. The virulence
functions of
the Agrobacterium tumefaciens host will direct the insertion of the construct
and
adjacent marker into the plant cell DNA when the cell is infected by the
bacteria using
binary T DNA vector (Bevan (1984) Nuc. Acid Res. 12:8711-8721) or the
co-cultivation procedure (Horsch et al (1985) Science 227:1229-1231).
Generally, the
Agrobacterium transformation system is used to engineer dicotyledonous plants
(Bevan et al (1982) Ann. Rev. Genet 16:357-384; Rogers et al (1986) Methods
Enrymol. 118:627-641 ). The Agrobacterium transformation system may also be
used
to transform, as well as transfer, DNA to monocotyledonous plants and plant
cells.
(see Hernalsteen et al (1984) EMBOJ3:3039-3041; Hooykass-Van Slogteren et al
(1984) Nature 311:763-764; Grimsley et al (1987) Nature 325:1677-179; Boulton
et
al (1989) Plant Mol. Biol. 12:31-40.; and Gould et al (1991) Plant Physiol.
95:426-434).
Alternative gene transfer and transformation methods include, but are not
limited to, protoplast transformation through calcium-, polyethylene glycol
(PEG)- or
electroporation-mediated uptake of naked DNA (see Paszkowski et al. (1984)
EMBO
J3:2717-2722, Potrykus et al. (1985) Molec. Gen. Genet. 199:169-177; Fromm et
al.
(1985) Proc. Nat. Acad. Sci. USA 82:5824-5828; and Shimamoto (1989) Nature
338:274-276) and electroporation of plant tissues (D'Halluin et al. (1992)
Plant Cell
4:1495-1505). Additional methods for plant cell transformation include
microinjection, silicon carbide mediated DNA uptake (Kaeppler et al. (1990)
Plant
Cell Reporter 9:415-418), and microprojectile bombardment (see Klein et al.
(1988)
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Proc. Nat. Acad. Sci. USA 85:4305-4309; and Gordon-Kamm et al. (1990) Plant
Cell
2:603-618).
Transformed plant cells which are produced by any of the above
transformation techniques can be cultured to regenerate a whole plant which
possesses
the transformed genotype and thus the desired phenotype. Such regeneration
techniques rely on manipulation of certain phytohormones in a tissue culture
growth
medium, typically relying on a biocide and/or herbicide marker which has been
introduced together with the desired nucleotide sequences. Plant regeneration
from
cultured protoplasts is described in Evans, et al., "Protoplasts Isolation and
Culture" in
Handbook of Plant Cell Culture, pp. 124-176, Macmillian Publishing Company,
New
York, 1983; and Binding, Regeneration of Plants, Plant Protoplasts, pp. 21-73,
CRC
Press, Boca Raton, 1985. Regeneration can also be obtained from plant callus,
explants, organs, pollens, embryos or parts thereof. Such regeneration
techniques are
described generally in Klee et al (1987) Ann. Rev. ofPlant Phys. 38:467-486.
The nucleic acids of the invention can be used to confer desired traits on
essentially any plant. A wide variety of plants and plant cell systems may be
engineered for the desired physiological and agronomic characteristics
described
herein using the nucleic acid constructs of the present invention and the
various
transformation methods mentioned above. In preferred embodiments, target
plants
and plant cells for engineering include, but are not limited to, those
monocotyledonous and dicotyledonous plants, such as crops including grain
crops
(e.g., wheat, maize, rice, millet, barley), fruit crops (e.g., tomato, apple,
pear,
strawberry, orange), forage crops (e.g., alfalfa), root vegetable crops (e.g.,
carrot,
potato, sugar beets, yam), leafy vegetable crops (e.g., lettuce, spinach);
flowering
plants (e.g., petunia, rose, chrysanthemum), conifers and pine trees (e.g.,
pine fir,
spruce); plants used in phytoremediation (e.g., heavy metal accumulating
plants); oil
crops (e.g., sunflower, rape seed) and plants used for experimental purposes
(e.g.,
Arabidopsis). Thus, the invention has use over a broad range of plants,
including, but
not limited to, species from the genera Asparagus, Avena, Brassica, Citrus,
Citrullus,
Capsicum, Cucurbita, Daucus, Glycine, Hordeum, Lactuca, Lycopersicon, Malus,
Manihot, Nicotiana, Oryza, Persea, Pisum, Pyrus, Prunus, Raphanus, Secale,
Solanum, Sorghum, Triticum, Vitis, Vigna, and Zea.
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One of skill in the art will recognize that after the expression cassette is
stably
incorporated in transgenic plants and confirmed to be operable, it can be
introduced
into other plants by sexual crossing. Any of a number of standard breeding
techniques can be used, depending upon the species to be crossed.
A transformed plant cell, callus, tissue or plant may be identified and
isolated
by selecting or screening the engineered plant material for traits encoded by
the
marker genes present on the transforming DNA. For instance, selection may be
performed by growing the engineered plant material on media containing an
inhibitory amount of the antibiotic or herbicide to which the transforming
gene
construct confers resistance. Further, transformed plants and plant cells may
also be
identified by screening for the activities of any visible marker genes (e.g.,
the
(3-glucuronidase, luciferase, B or C 1 genes) that may be present on the
recombinant
nucleic acid constructs of the present invention. Such selection and screening
methodologies are well known to those skilled in the art.
Physical and biochemical methods also may be used to identify plant or plant
cell transformants containing the gene constructs of the present invention.
These
methods include but are not limited to: 1) Southern analysis or PCR
amplification for
detecting and determining the structure of the recombinant DNA insert; 2)
Northern
blot, S 1 RNase protection, primer-extension or reverse transcriptase-PCR
amplification for detecting and examining RNA transcripts of the gene
constructs; 3)
enzymatic assays for detecting enzyme or ribozyme activity, where such gene
products are encoded by the gene construct; 4) protein gel electrophoresis,
Western
blot techniques, immunoprecipitation, or enzyme-linked immunoassays, where the
gene construct products are proteins. Additional techniques, such as in situ
hybridization, enzyme staining, and immunostaining, also may be used to detect
the
presence or expression of the recombinant construct in specific plant organs
and
tissues. The methods for doing all these assays are well known to those
skilled in the
art.
Effects of gene manipulation using the methods of this invention can be
observed by, for example, northern blots of the RNA (e.g., mRNA) isolated from
the
tissues of interest. Typically, if the amount of mRNA has increased, it can be
assumed that the endogenous dwf4 gene is being expressed at a greater rate
than
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before. Other methods of measuring DWF4 activity can be used. For example,
cell
length can be measured at specific times. Because dwf4 affects the BR
biosynthetic
pathway, an assay that measures the amount of BL can also be used. Such assays
are
known in the art. Different types of enzymatic assays can be used, depending
on the
substrate used and the method of detecting the increase or decrease of a
reaction
product or by-product. In addition, the levels of DWF4 protein expressed can
be
measured immunochemically, i.e., ELISA, RIA, EIA and other antibody based
assays
well known to those of skill in the art, by electrophoretic detection assays
(either with
staining or western blotting), and sterol (BL) detection assays.
The transgene may be selectively expressed in some tissues of the plant or at
some developmental stages, or the transgene may be expressed in substantially
all plant tissues, substantially along its entire life cycle. However, any
combinatorial
expression mode is also applicable.
The present invention also encompasses seeds of the transgenic plants
1 S described above wherein the seed has the transgene or gene construct. The
present
invention further encompasses the progeny, clones, cell lines or cells of the
transgenic
plants described above wherein said progeny, clone, cell line or cell has the
transgene
or gene construct.
Polypentides
The present invention also includes DWF4 polypeptides, including such
polypeptides as a fusion, or chimeric protein product (comprising the protein,
fragment, analogue, mutant or derivative joined via a peptide bond to a
heterologous
protein sequence (of a different protein)). Such a chimeric product can be
made by
ligating the appropriate nucleic acid sequences encoding the desired amino
acid
sequences to each other by methods known in the art, in the proper coding
frame, and
expressing the chimeric product by methods commonly known in the art.
As noted above, DWF4 phenotype includes any macroscopic, microscopic or
biochemical changes which are characteristic of over- or under-expression of
dwf4.
Thus, DWF4 polypeptide phenotype (e.g., activities) can include any activity
that is
exhibited by the native DWF4 polypeptide including, for example, in vitro, in
vivo,
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biological, enzymatic, immunological, substrate binding activities, etc. Non-
limiting
examples of DWF4 activities include:
(a) activities displayed by other heme-thiolate enzymes;
(b) characteristic Soret absorption peak at 40 nm when the substrate-bound
reduced form is exposed to the lights (see, e.g., Jefcoate et al., infra);
(c) hydroxylation of various substrates via monooxygenase activity, which
utilizes molecular oxygen and reducing equivalents from NAD(P)H;
(d) oxidation, dealkylation, deaminoation, dehalogenation, and sulfoxide
formation that are involved in a variety of biological events in plants and
animals
(e.g., catabolism, anabolism, and xenobiotic activities);
(e) recognition of at two substrates: campestanol (CN) and 6-
deoxocastasterone (6-deoxoCS);
(f) 22a-hydroxylase activity;
(g) DWF4 phenotypic activities such as modulation of cell length, periods of
flowering, branching, seed production, leaf size, and sterol composition in a
plant;
(h) regulation of gibberellic acid, cytokinins and/or auxin;
(i) induce resistance to plant pathogens (see, e.g., U.S. Patent No.
5,952,545);
(j) accelerating growth at low temperatures; and
(k) accelerating growth in dark conditions.
A DWF4 analog, whether a derivative, fragment or fusion of native DWF4
polypeptides, is capable of at least one DWF4 activity. Preferably, the
analogs exhibit
at least 60% of the activity of the native protein, more preferably at least
70% and
even more preferably at least 80%, 85%, 90% or 95% of at least one activity of
the
native protein.
Further, such analogs exhibit some sequence identity to the native DWF4
polypeptide sequence. Preferably, the variants will exhibit at least 35%, more
preferably at least 59%, even more preferably 75% or 80% sequence identity,
even
more preferably 85% sequence identity, even more preferably, at least 90%
sequence
identity; more preferably at least 95%, 96%, 97%, 98% or 99% sequence
identity.
DWF4 analogs can include derivatives with increased or decreased activities
as compared to the native DWF4 polypeptides. Such derivatives can include
changes
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within the domains, motifs and/or consensus regions of the native DWF4
polypeptide,
which are described in detail in Example 3.
Once class of analogs is those polypeptide sequences that differ from the
native DWF4 polypeptide by changes, insertions, deletions, or substitution; at
positions flanking the domain and/or conserved residues. For example, an
analog can
comprise (1) the domains of a DWF4 polypeptide and/or (2) residues conserved
between the DWF4 polypeptide and other cytochrome P450 proteins, for example
as
shown in Figure 3 and described in Example 3.
Another class of analogs includes those that comprise a DWF4 polypeptide
sequence that differs from the native sequence in the domain of interest or
conserved
residues by a conservative substitution. For example, an analog that exhibits
increased sterol binding can have optimized sterol binding domain sequences
that
differ from the native sequence.
Yet another class of analogs includes those that lack one of the in vitro
activities or structural features of the native DWF4 polypeptides, for
example,
dominant negative mutants or analogs that comprise a heme-binding domain but
contain an inactivated steroid binding domain.
DWF4 polypeptide fragments can comprise sequences from the native or
analog sequences, for example fragments comprising one or more of the
following
P450 domains or regions: A, B, C, D, anchor binding, and proline rich. Such
domains
and regions are shown in Figures 2B, 3 and described in Example 3.
Fusion polypeptides comprising DWF4 polypeptides (e.g., native, analogs, or
fragments thereof) can also be constructed. Non-limiting examples of other
polypeptides that can be used in fusion proteins include chimeras of DWF4
polypeptides and fragments thereof; and P450 polypeptides or fragments
thereof, such
as those shown in Figure 3.
In addition, DWF4 polypeptides, derivatives (including fragments and
chimeric proteins), mutants and analogues can be chemically synthesized. See,
e.g.,
Clark-Lewis et al. (1991) Biochem. 30:3128-3135 and Mernfield (1963) J. Amer.
Chem. Soc. 85:2149-2156. For example, DWF4, derivatives, mutants and analogues
can be synthesized by solid phase techniques, cleaved from the resin, and
purified by
preparative high performance liquid chromatography (e.g., see Creighton, 1983,
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Proteins, Structures and Molecular Principles, W. H. Freeman and Co., N.Y.,
pp.
50-60). DWF4, derivatives and analogues that are proteins can also be
synthesized by
use of a peptide synthesizer. The composition of the synthetic peptides may be
confirmed by amino acid analysis or sequencing (e.g., the Edman degradation
procedure; see Creighton, 1983, Proteins, Structures and Molecular Principles,
W. H.
Freeman and Co., N.Y., pp. 34-49).
Further, the dwf4 polynucleotides and DWF4 polypeptides described herein
can be used to generate antibodies that specifically recognize and bind to the
protein
products of the dwf4 polynucleotides. (See, Harlow and Lane, eds. (1988)
"Antibodies: A Laboratory Manual"). The DWF4 polypeptides and antibodies
thereto
can also be used in standard diagnostic assays, for example,
radioimmunoassays,
ELISA (enzyme linked immunoradiometric assays), "sandwich" immunoassays,
immunoradiometric assays, in situ immunoassay, western blot analysis,
immunoprecipitationassays, immunofluorescent assays and PAGE-SDS.
Applications
The present invention finds use in various applications, for example,
including
but not limited to those listed above.
The polynucleotide sequences may additionally be used to isolate mutant dwf4
gene alleles. Such mutant alleles may be isolated from plant species either
known or
proposed to have a genotype which contributes to altered plant morphology.
Additionally, such plant dwf4 gene sequences can be used to detect plant dwf4
gene
regulatory (e.g., promoter or promoter/enhancer) defects which can affect
plant
growth.
The molecules of the present invention can be used to provide plants with
increased seed and/fruit production, extended flowering periods and increased
branching. The molecules described herein can be used to alter the sterol
composition
of a plant, thereby increasing or reducing cholesterol content in the plant. A
still
further utility of the molecules of the present invention is to provide a tool
for
studying the biosynthesis of brassinosteriods, both in vitro and in vivo.
The dwf4 gene of the invention also has utility as a transgene encoding a
cytochrome P450 protein that mediates multiple 22a hydroxylation steps in
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brassinosteriod biosynthesis which results in a transgenic plant to alter
plant structure
or morphology. The dwf4 gene also has utility for encoding the DWF4 protein in
recombinant vectors which may be inserted into host cells to express the DWF4
protein. Further, the dwf4 polynucleotides of the invention may be utilized
(1) as
nucleic acid probes to screen nucleic acid libraries to identify other
enzymatic genes
or mutants; (2) as nucleic acid sequences to be mutated or modified to produce
DWF4
protein variants or derivatives; (3) as nucleic acids encoding 22a-hydroxylase
in
molecular biology techniques or industrial applications commonly known to
those
skilled in the art.
The dwf4 nucleic acid molecules may be used to design plant dwf4 antisense
molecules, useful, for example, in plant dwf4 gene regulation or as antisense
primers
in amplification reactions of plant dwf4 gene nucleic acid sequences. With
respect to
plant dwf4 gene regulation, such techniques can be used to regulate, for
example,
plant growth, development or gene expression. Further, such sequences may be
used
as part of ribozyme and/or triple helix sequences, also useful for dwf4 gene
regulation.
The dwf4 control element (e.g., promoter) of the present invention may be
utilized as a plant promoter to express any protein, polypeptide or peptide of
interest
in a transgenic plant. In particular, the dwf4 promoter may be used to express
a
protein involved in brassinosteriod biosynthesis.
The Arabidopsis DWF4 protein of the invention can be used in any
biochemical applications (experimental or industrial) where 22a-hydroxylase
activity
is desired, for example, but not limited to, regulation of BL synthesis,
regulation of
other sterol synthesis, modification of elongating plant structures, and
experimental or
industrial biochemical applications known to those skilled in the art.
EXPERIMENTAL
Below are examples of specific embodiments for carrying out the present
invention. The examples are offered for illustrative purposes only, and are
not
intended to limit the scope of the present invention in any way.
Efforts have been made to ensure accuracy with respect to numbers used (e.g.,
amounts, temperatures, etc.), but some experimental error and deviation
should, of
course, be allowed for.
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Example 1: Materials and Methods
A. Plant Growth Conditions
The conditions used for plant growth were essentially as described previously
(Feldmann (1991) Plant J. 1:71-82 ; Forsthoefel et al. (1992) Aust. J. Plant
Physiol.
19:353-366), except that agar-solidified medium contained 0.5% sucrose.
Seedlings
up to 2 weeks of age (6 weeks of age for dark-growth experiments) were grown
on
0.8% agar-solidified medium containing 1 x Murashige and Skoog 1962 salts
(Murashige, T., and Skoog, F. (1962) Physiol. Plant. 15:473-497) and 0.5%
sucrose
(w/v) and cold treated (4°C) for 2 days in the dark before transfer to
the light (24 hr
light; 80 ~mol m z sec-' ); older plants were grown in potting soil. The
plates were
sealed with Parafilm (American National Can Co., Chicago, IL) for the entire
experiment. For nucleic acid extraction, genetic analysis, and other
experiments in
which mature plants were required, seeds were sown on Metromix 350 (Grace
Sierra,
Milpitas, CA) presoaked with distilled water. The pots were covered with
plastic
wrap and cold treated (4°C) for two days before transfer to a growth
chamber (16:8,
light [240 ~mol m Z sec']:dark; 22 and 21 °C, respectively, and 75 to
90% humidity).
The plastic wrap was removed 5 days after germination, and the pots were
subirrigated in distilled water as required. Germination of seeds for dark
growth
experiments was induced by overnight exposure of the seeds to light
immediately
after removing the plates from incubation at 4°C. The dwf4-1 and dwf4-2
mutations
were in the Arabidopsis thaliana ecotype Wassilewskija (Ws-2) background; the
dwf4-3 and dwf4-4 mutations were in the Enkheim (En-2) background.
B. Analytical Methods
Protoplasts were obtained by overnight incubation of sliced leaves in 0.1
cellulysin, 0.1 % driselase, 0.1 % macerase (Calbiochem, San Diego, CA) in 125
mM
Mes, pH 5.8, 0.5 M mannitol, and 7 mM CaCl2 (Galbraith et al. (1992) Planta
186:324-326 . Immediately before observation, chloroplasts were stained with a
solution of 1.5% KI and 1% I,. Measurements were performed as described for
tissue
sections, and plane areas were calculated according to the formula A = ~rz.
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Chlorophyll determinations were performed from 2-week-old soil-grown
plants. Green tissue was weighed, frozen in liquid nitrogen, and extracted in
dim light
with 80% acetone in the presence of a mixture of equal parts sand, NaHC03, and
Na,S04. After brief centrifugation, the supernatant was collected and the
extraction
was repeated twice, pooling the supernatants from each sample. Chlorophylls a
and b
were measured spectrophotometrically, as described in Chory et al. ( 1991 ),
supra.
C. Growth Signal Response Measurements
Gibberellic acid (GA) response was assayed on plants grown individually in
5.7-cm pots. Once inflorescences reached 1 to 2 mm in height, they were
sprayed
weekly with 1 mM GA3 (Sigma). Control plants were sprayed with water. One week
after the third spraying, plants were collected, and the length of the main
stem was
measured between the top of the rosette and the base of the most distal
pedicel; 13 to
18 plants of each line were measured per treatment. Auxin response was tested
by
growing seedlings for 10 days under 16 hr of light on vertically oriented agar
plates
containing various concentrations of 2,4-D (Gibco, Grand Island, NY). Genetic
interaction with the hy2 mutation was tested by growing seedlings under
continuous
light for 7 days. Brassinolide (BL) response was determined in liquid culture,
as
described by Clouse et al. (1993), supra, except that three or four seedlings
were
grown in each well of a 24-well culture plate for 7 days. Measurements were
taken for
10 to 20 seedlings for each genotype and condition, under a dissection
microscope
fitted with an ocular micrometer.
D. Microscopy
Tissues were fixed in 2% glutaraldehyde and 0.05 M sodium cacodylate, pH
6.9, for 2 hr at room temperature or overnight at 4°C, followed by
three washes in
buffer. For light microscopy, 1% safranin was included in the first wash, and
embedding was performed in Paraplast Plus (Oxford Labware, St. Louis, MO).
Ten-millimeter sections from five individual plants per line were analyzed and
photographed, and cell measurements were taken using a ruler on S x 7 inch
prints. A
print of a hemocytometer grid at the same final magnification was used for
calibration. At least 25 cells were measured per sample, with a minimum of 150
cells
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per line. For electron microscopy, the tissues were treated after fixation
with 1%
tannic acid in buffer for 30 min, washed three times, and postfixed in 1 %
Os04 in
buffer for 2 hr, followed by five washes and dehydration through an ethanol
series.
Samples for transmission electron microscopy were embedded in Spurr's resin.
Sections (90 nm) were stained with saturated uranyl acetate followed by
Reynolds's
lead citrate (Reynolds (1963) J. Cell Biol. 17:208-212) and examined in a JEOL
(Tokyo, Japan) 100-CX instrument. For scanning electron microscopy, samples
were
transferred to freon 113, critical point dried, and sputter-coated with 30 to
50 nm of
gold. Analysis was performed in a microscope (ISI model DS 130; Topcon, Inc.,
Paramus, NJ) with an accelerating voltage of 15 kV. Electron microscopy was
performed at the Electron Microscope Facility, Division of Biotechnology,
Arizona
Research Laboratories, University of Arizona.
Example 2: Isolation of dwf4 gene
A. Isolation of the DWF4 Gene
The dwf4-1 mutation was identified in a screen of 14,000 transformants of
Arabidopsis, resulting in a dwarfed phenotype similar to dwfl (Feldmann and
Marks
(1987) Mol. Gen. Genet. 208:1-9; Feldmann et al. (1989) Science 243:1351-1354;
referred to as diminuto in Takahashi et al. (1995) Genes Dev. 9:97-107 and
Szekeres
et al., supra) and det2 (Azpiroz et al. (1998), supra). Two independent lines
were
found that segregated for a similar phenotype: both were shorter than dwfl,
but their
rosette diameter was comparable to that mutant. These dwarfs were also
essentially
infertile. The most striking aspect of the morphology of these mutants is
their
similarity to det2 (Chory et al. (1991) Plant Cell 3:445-459). For this
reason, further
analysis was conducted with these lines. After being found to be allelic to
each other,
both were designated as dwf4.
dwf4-1 segregated for a single kanamycin resistance marker, and gel blot
analysis with DNA from single plants of this family confirmed that the pattern
is
consistent with a single insert. The dwf4 mutation was subsequently shown to
be
inherited as a monogenic, recessive Mendelian trait that, in dwf4-1,
cosegregates with
the dominant kanamycin resistance marker contained in the T-DNA, suggesting
that
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the mutation in this line may be a disrupted, tagged allele. dwf4-2 also
contains a
single kanamycin resistance marker, but it failed to cosegregate with the
dwarf
phenotype. Two additional alleles (dwf4-3 and dwf4-4) were identified among
dwarf
mutants obtained from the Nottingham Arabidopsis Resource Centre (Nottingham,
UK; N365 and N374). Unless otherwise indicated, all experiments presented
below
were performed with dwf4-1.
Standard molecular techniques were performed as described previously
(Sambrook et al. 1989). The plant DNA flanking the T-DNA was cloned using the
plasmid rescue technique as described by Dilkes and Feldmann (1998) "Cloning
genes from T-DNA tagged mutants" in Methods in Molecular Biology: Arabidopsis
Protocol, J. Martinez-Zapater and J. Saunas, eds (Totowa, NJ: Humana Press),
pp.
339-351. Briefly, dwf4-1 genomic DNA was digested with EcoRI (for the right
border) or SaII (for the left border), ligated under conditions to maximize
intramolecular events, and introduced into competent Escherichia coli cells.
The
resulting colonies were screened on ampicillin. Five colonies from the left
border
transformation contained plant DNA flanking the insertion site. The
restriction
pattern displayed two different types of plant DNA. Three contained a 5.6-kb
insert,
whereas the other two contained a 1.1-kb insert. This result suggested that
the
T-DNA insert in dwf4-1 was flanked by two left border sequences. The existence
of
two left border sequences was confirmed by gel blot analysis with genomic DNA,
using the putative plant flanking DNAs as probes. A single wild-type EcoRI
fragment
was split into two fragments in dwf4-1.
Wild-type genomic clones were isolated from a library made from Ws-2
DNA by using the 5.6-kb fragment as a probe. The library was constructed using
~,
DASH-II arms (Stratagene, La Jolla, CA). Approximately 10,000 primary plaques
were screened. Duplicate-filter screening resulted in 12 positives.
Restriction
mapping of the secondary clones revealed that some contained part of the DWF4
locus. In fact, one of the clones, D4G12-1, contained an intact 13-kb DNA
spanning
the T-DNA insertion site. The 13-kb insert in D4G12-1 was subcloned into
pBluescript SK- (Stratagene). Subclones were sequenced from each end of the
insert
by using the universal primers in the plasmid. DNA sequencing was performed
using
an ABI 377 (Perkin-Elmer, Norwalk, CT ) automated sequencer at the Arizona
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Research Laboratories (Tucson, AZ).
Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to isolate
a cDNA clone. RNA was isolated from 5-day-old dark- and light-grown seedlings.
Superscript II reverse transcriptase (BRL, Gaithersburg, MD) was used for the
cDNA
synthesis, according to the manufacturer's protocol. Briefly, 7 pg of total
RNA was
mixed with the reverse primer, D4R3. To the heat-denatured RNA-primer mix, the
RT
mixture was added and incubated for 1 hr at 43 ° C. Two microliters of
RT product
was used for PCR amplification by using different primers sets intended to
cover all
of the putative coding region. RT-PCR products were fractionated on an 0.8%
agarose gel (Sambrook et al. 1989); the expected bands were purified using a
Geneclean kit (BIO 101, Inc., Vista, CA), further amplified, and sequenced to
determine the coding region.
S. Sequencing
dwf4-2 was isolated from a T-DNA mutant population as an untagged allele,
whereas dwf4-3 and dwf4-4 were obtained from plants obtained from the
Nottingham
Arabidopsis Stock Centre (University of Nottingham, UK; stock nos. N365 and
N374); the mutagenesis method for these two lines is not known. Based on the
DNA
sequence of wild-type genomic DNA, pairs of primers were designed to amplify
~ 1-kb stretches of genomic DNA. Oligonucleotide sequences are shown S' to 3'.
The
numbers shown correspond to positions in the genomic sequence, with the
adenine
base in the translation initiation codon set as position 1. D40VERF,
1-ATGTTCGAAACAGAGCATCATACT-24 (SEQ ID N0:3); D4PRM,
(-1)-CCTCGATCAAAGAGAGAGAGA-(-21) (SEQ ID N0:4); D4RTF,
143-TTCTTGGTGAAACCATCGGTTATCTTAAA-171 (SEQ ID NO:S); D4RTR,
853-TATGATAAGCAGTTCCTGGTAGATTT-828 (SEQ ID N0:6); D4F1,
(-242)-CGAGGCAAC-AAAAGTAATGAA-(-222) (SEQ ID N0:7); D4R1,
689-GTTAGAAACTCTAAAGATTCA-669 (SEQ ID N0:8); D4F2,
576-GATTCTTGGCAACAAAACTCTAT-598 (SEQ ID N0:9); D4R2,
1685-CCGAACATCTTTGAGTGCTT-1666 (SEQ ID NO:10); D4F3,
1606-GTGTGAAGGTTATAAATGAAACTCTT-1631 (SEQ ID NO:11); D4R3,
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3156-GGTTTAATAGTGTCGACACTAATA-3132 (SEQ ID N0:12); D4F4,
2316-CCGATGACTTGTACGTGCGTTA-2337 (SEQ ID N0:13); D4F5,
730-GCGAAGCATATAATGAGTATGGAT-753 (SEQ ID N0:14); and D4R5,
1876-GTTGGTCATAACGAGAATTATCCAAA-1851 (SEQ ID NO:15). Because
the two stock center lines were in a different genetic background than the
wild-type
gene that we had sequenced (WS), primers were based primarily on the exon
sequence
to avoid sequence variation between introns. Genomic DNA isolated from the
mutants was subjected to PCR, using these primer sets. The amplified DNA
fragments were fractionated on 0.8% TAE agarose gel (Sambrook et al. 1989),
purified using Geneclean (BIO 101, Inc.) or QiaquickTM columns (Qiagen Inc.,
Chatsworth, CA), and sequenced. Putative mutations were identified by
comparing
the mutant DNA sequence with the wild-type sequence. The sequence was
confirmed
by sequencing independently amplified fragments at least three times for each
mutation to eliminate PCR misincorporation.
C. Sequence Analysis
Annotations in multiple sequence alignment were performed using the
ALSCRIPT package provided by Barton, G.J. (1993) Protein Eng. 6:37-40.
Searches
for similar protein sequence were performed with the BLAST program (Altschul
et al.
(1990), supra). In addition, useful packages, available on the Internet, such
as
promoter, protein targeting, polyadenylation site, and splice site, have been
employed
to characterize the DNA and protein sequence (consolidated in the search
launcher,
Baylor College of Medicine, Baylor, TX). All other sequence analysis was
performed
using the Genetics Computer Group (Madison, WI) software package.
Analysis of the complete genomic sequence, starting at the EcoRI site, with
the promoter prediction by neural network (NNPP) package
(http://www-hgc.lbl.gov./projects/promoter.html), indicated that the gene
included a
putative promoter (TATAT is found in the putative promoter region between
nucleotides -143 to -78) and polyadenylation signal sequences (AATAA near a
position at 3238 by and a putative GU-rich signature from 3283 to 3290 bp).
Unsuccessful attempts to detect mRNA by tissue-specific RNA gel blot
analysis, using the 4.8-kb fragment as a probe, suggested that DWF4 encoded a
rare
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message. In addition, there were no matching expressed sequence tags in the
Arabidopsis database. Therefore, we screened two different cDNA libraries made
with either normalized mRNA from different tissues or RNA from floral tissues,
using the 4.8-kb fragment as a probe (ABRC stock numbers CD4-7 and CD4-6,
respectively). After finding no positives in 109 clones screened, we chose to
directly
amplify DWF4 cDNA from total RNA made from 5-day-old seedlings, using reverse
transcriptase-polymerase chain reaction (RT-PCR). Whereas RNA from both
light-grown and dark-grown seedlings yielded the expected RT-PCR products, RNA
from dark-grown seedlings generated significantly more. The bands were gel
purified
and sequenced. Alignment of the genomic and cDNA sequences indicated that the
DWF4 gene was composed of eight exons and seven introns (Figure 2A; Figure
10).
- Sequence analysis of the dwf4-1 allele revealed that the T-DNA was inserted
in the 5' end of intron 7 (Figure 2A). In addition, sequence analysis of the
left border
plant junctions indicated that at one junction (5'), 75 by of unknown DNA was
inserted, whereas at the other junction (3'), 24 by of left border and 19 by
of plant
DNA were deleted. To prove that DWF4 had been cloned, two other dwf4 alleles
(dwf4-2 and dwf4-3) were sequenced to identify possible lesions. As shown in
Figure
2B, dwf4-2 contained a deletion of three conserved amino acids (324 to 326)
caused
by a 9-by deletion, and dwf4-3 contained a premature stop codon (289) caused
by
changing a tryptophan codon (UGG) to a nonsense codon (UGA). Due to a
premature
stop codon, translation is predicted to be terminated before the heme binding
domain,
which is essential for cvtochrome P450 function (Poulos et al. (1985) J. Biol.
Chem.
260:16122-16130). Because T-DNA-generated alleles dwf4-l and dwf4-2 and an
additional mutant allele all possess loss-of function mutations affecting the
same
protein, we conclude that we have cloned the DWF4 gene.
Example 3: The DWF4 Gene Encodes a Cytochrome P450
The open reading frame of DWF4 encodes a protein composed of 513 amino
acids. BLAST database searches (Altschul et al. (1990) J. Mol. Biol. 215:403-
410)
for similar sequences yielded a superfamily of cytochrome P450 proteins as
significant high-scoring segment pairs. Cytochrome P450s are heme-thiolate
enzymes. They display a characteristic Soret absorption peak at 450 nm when
the
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substrate-bound, reduced form is exposed to the light (Jefcoate (1978)
"Measurement
of substrate and inhibitor binding to microsomal cytochrome P-450 by
optical-difference spectroscopy" in Methods in Enzymology, Vol. 52, S.
Fleischer and
L. Packer, eds (London: Academic Press), pp. 258-279). Typical microsomal
cytochrome P450s hydroxylate various substrates via their monooxygenase
activity,
which utilizes molecular oxygen and reducing equivalents from NAD(P)H. In
addition to the hydroxylation, other activities of cytochrome P450 enzymes,
such as
oxidation, dealkylation, deamination, dehalogenation, and sulfoxide formation,
are
involved in a variety of biological events in catabolism, anabolism, and
xenobiotic
metabolism in plants as well as animals (reviewed in West (1980)
"Hydroxylases,
monooxygenases, and cytochrome P-450" in The Biochemistry of Plants: A
Comprehensive Treatise, Vol. 2, Metabolism and Respiration, D.D. Davies, ed
(New
York: Academic Press), pp. 317-365; Nebert and Gonzalez (1987), supra;
Guengerich
(1990) Crit. Rev. Biochem. Mol. Biol. 25:97-152, Guengerich (1993) Am. Sci.
81:440-447; Durst (1991) "Biochemistry and physiology of plant cytochrome P-
450"
in Microbial and Plant Cytochromes P-450: Biochemical Characteristics, Genetic
Engineering and Practical Implications, K. Ruckpaul and H. Rein, eds (London:
Taylor and Francis), pp. 191-232; Bolwell et al. (1994) Phytochemistry
37:1491-1506; Durst and Nelson (1995), supra; Schuler (1996) CRC Crit. Rev.
Plant
Sci. 15:235-284). Evolutionarily, cytochrome P450s have been found in a broad
spectrum of living organisms, and they share significant homology at the amino
acid
sequence level. Thus, it has been proposed that all known cytochrome P450s
were
derived from a common ancestor (Nelson and Strobel (1987) Mol. Biol. Evol.
4:572-593).
Typical cytochrome P450s contain four characteristic domains as defined by
Kalb and Loper 1988. Of the four domains, A, B, C, and D, at least two of them
have
been assigned specific functions. Domain A binds a substrate and molecular
oxygen,
and domain D has been shown to bind heme-prosthetic groups via a thiolate bond
(Poulos et al. 1985). Thus, typically, microsomal cytochrome P450 enzymes can
be
identified by their characteristic signature sequences, including the heme
binding
domain, domain A (also referred to as dioxygen binding), domain B (steroid
binding),
and domain C (Nebert and Gonzalez (1987) Annu. Rev. Biochem. 56:945-993; Kalb
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and Loper (1988) Proc. Natl. Acad. Sci. USA 85:7221-7225). All of these
signature
sequences were found in DWF4; the relative positions of the domains are
indicated in
Figure 2B.
Durst and Nelson (1995) Drug Metab. Drug Interact. 12:189-206 classified
plant cytochrome P450s into two distinct groups based on their clustering
nature in a
phylogenetic tree. All of the group A families cluster and are assumed to
originate
from a common plant P450 ancestor. The group A cytochrome P450s conform to the
characteristic consensus sequences (A/G)GX(D/E)T(T/S) in domain A (also called
helix I) and PFG(A/S/V)GRRXC(P/A/V)G of the heme binding domain (D) with only
a few exceptions. Group A cytochrome P450s appear to catalyze plant-specific
reactions such as lignin biosynthesis (Figure 6; GenBank accession number
P48421).
By contrast, P450s that do not belong to group A (non-A P450s) are scattered
in the
phylogenetic tree. They share more amino acid identity/similarity with P454s
found
in animals, microbes, and fungi than with those found in plants. The non-A
P450s
possess functions, such as steroid metabolism, that are not limited to plants.
Generally, non-A P450s have limited homology with known domains described for
group A.
The most similar protein to DWF4 is the Arabidopsis CPD protein, a non-A
P450. A mutation in CPD also caused dwarfism (Szekeres et al. 1996; CYP90A1,
GenBank accession number X87368). DWF4 and CPD share 43% identity and 66%
similarity. Conforming to the recommended nomenclature for cytochrome P450
enzymes, DWF4 and CPD (CYP90A1) are grouped into the same family within
different subgroups (Durst and Nelson (1995) Drug Metab. Drug Interact.
12:189-206). As such, DWF4 represents a second member of the CYP90 family and
is designated CYP90B 1. Sequence similarity between the two proteins occurs
throughout their length, with the greatest similarity in the classically
conserved
domains. Residues conserved between DWF4 and CYP90A are boxed and italicized
in Figure 3. The second most similar protein is the tomato CYP85 (Bishop et
al.
(1996), supra; GenBank accession number U54770). A mutation in this gene also
results in dwarfism. DWF4 and CYP85 share 35% identity and 59% similarity in
their
overall protein sequences.
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Six cytochrome P450 sequences with the greatest homology to DWF4,
CYP90A1, CYP85, CYP88 (Winkler and Helentjaris (1995) Plant Cell 7:1307-1317;
GenBank accession number U32579), cyanobacteria CYP120 (Kaneko et al. (1996)
DNA Res. 3:109-136; GenBank accession number D64003), human CYP3A3X
(Molowa et al. (1986) Proc. Natl. Acad. Sci. USA 83:5311-5315; GenBank
accession
number M13785), and zebrafish CYP26 (White et al. White (1996) .l. Biol. Chem.
271:29922-29927; GenBank accession number U68234), were chosen for multiple
sequence alignment. Putative domains defined by Kalb and Loper (1988), supra
are
boxed and labeled in Figure 3. First, the heme binding domain pFGgFpRICpGke1
matches completely the sequence defined previously. Uppercase letters in the
domain
indicate amino acids conserved at all seven sequences in the alignment, and
lower-case letters represent residues conserved in at least half of the
proteins. Of the
amino acids conserved in the heme binding domain, the function of the
cysteinyl is
established as a thiolate ligand to the heme (Poulos et al. (1985), supra).
Domain A is defined by xllfaGhEttssxIxxa. Lowercase x's indicate variable
amino acids. An invariant glutamate (E) preceded threonine (T) at position
314,
T314, which is believed to bind dioxygen, was conserved in all proteins
compared
except CYP88 of maize. The second signature sequence, domain B, is also
conserved
in DWF4 with significant similarity. A valine at position 370 is conserved in
all of
the proteins, but it does not appear in Kalb and Loper's classic report (1988)
on
conserved domains. Again, DWF4 matches the domain C consensus sequence.
Finally, the anchoring domain in the N-terminal end was distinguished by a
repeat of
the hydrophobic residue leucine. In addition, in DWF4, two acidic (glutamate)
and
two basic (histidine) residues precede the repeated leucine in the N-terminal
leader
sequence. These charged residues may add more stability to the membrane
topology
of the protein as a strong start-stop transfer peptide (von Heijne (1988)
Biochim.
Biophys. Acta 947:307-333).
Thus, phylogenetic analyses of these seven proteins with cytochrome P450s
unique to plants (group A; Durst and Nelson (1995), supra) indicate that DWF4
does
not cluster with these cytochrome P450s (Figure 6). Rather, DWF4 clustered
with
cytochrome P450s from other organisms: cyanobacteria (CYP120), rat (CYP3A2),
human (CYP3A3X), and plants (CYP90, CYP85, and CYP88). DWF4 also deviates
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from the consensus sequence in the group A heme binding domain in that it
possesses
a PFGGGPRLCAG sequence in which arginine (R) is substituted for proline (P).
However, domain A of DWF4, AGHETS, fits the consensus of domain A of group A.
These characteristics suggest that DWF4 is a monooxygenase, similar to P450s
of
S group A, that utilizes molecular oxygen as a source of the hydroxyl group,
but it
mediates some reactions) that are not necessarily specific for plants, for
instance,
steroid hormone biosynthesis, which is a critical event for animals. In fact,
the
similarity of DWF4 to the rat testosterone 6~3-hydroxylase (34%; GenBank
accession
number 631895) or glucocorticoid-inducible hydroxylase (31%; Molowa et al.
1986;
GenBank accession number M13785) supports this idea. Further, the similarity
that
DWF4 shares with CYP90A and CYP85, 66 and 59%, respectively, is additional
proof that it is involved in plant steroid biosynthesis (Bishop et al. 1996 ;
Szekeres et
al. 1996).
Example 4: The dwf4 Phenotype
As formally defined, a plant with a dwarf phenotype is one that has a short,
robust stem and short, dark green leaves. dwf4 mutants are significantly
smaller than
the wild type and are dark green in color. They have short, rounded leaves.
Again, the
dwf4 phenotype is reminiscent of the light-regulatory mutant det2 (Chory et
al.,
supra); however, complementation analysis has shown that the two mutations are
not
allelic, with the dwf4 mutation mapping to the lower arm of chromosome 3 and
det2
mapping to chromosome 2 (Chory et al., supra). The results presented in Table
1
show that soil-grown dwf4 plants attained a height of <3 cm at 5 weeks,
whereas
wild-type plants grew to >25 cm. Moreover, individual organs, such as leaves,
were
invariably shorter in dwarf plants. dwf4 siliques were also markedly shorter
than those
of the wild type and were infertile. The loss of fertility of dwf4 was due to
the reduced
length of the stamen filaments relative to the gynoecium, which resulted in
mature
pollen deposition on the ovary wall rather than on the stigmatic surface. Hand
pollination of dwf4 flowers with either mutant or wild-type pollen resulted in
good
seed set without significantly changing the size of the siliques.
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Table 1: The Development
of Wild-Type and
dwf4-1 plants
Measurement Wild-Types dwf4-1 a
Five Weeks
Height 25.8 t 2.6 cm 2.8 ~ 0.3 cm
Leaf blade lengthb 1.72 ~ 0.36 cm 0.96 t 0.15 cm
Leaf blade widthb 0.77 ~ 0.10 cm 0.99 t 0.18
No. inflorescences 3.6 ~ 0.5 10.5 ~ 1.4
No. rosettes 7.1 ~ 0.9 13.5 ~ 1.3
Other
start of flowering 21.5 days 25.9 days
mature silique 1.16 ~ 0.07 cm 0.29 ~ 0 cm
length
No. seeds per 37.7 t 3.3 0.0
silique
Final no. of siliques336.5 t 90.6 988.4 ~ 214.2
Height at maturity 27.0 ~ 2.7 11.6 ~ 1.0 cm
" results shown are the average tSD of measurements taken from 10 plants
b measurements taken from the second pair of leaves
Another feature of dwf4 plants is a reduction in apical dominance, as was
evident by the threefold increase in the number of inflorescences at 5 weeks
of age
(Table 1). Mutants also had twice the number of rosette leaves, which may be
explained by a prolonged vegetative phase in the dwf4 plants. Development of
flowers
on the primary inflorescence was delayed by ~4 days in dwf4, but the flowering
phase
was significantly longer in the mutant, with senescence of the last flower
occurring at
---98 days compared with ~57 days for the wild type. One result of this delay
in
senescence was that dwf4 plants contained almost three times the number of
siliques
as did the wild type (Table 1).
The reduced stature observed in soil-grown dwf4 was also observed in
hypocotyls of agar-grown seedlings. Measurements of hypocotyl length over time
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indicated that not only were dwf4 seedlings shorter than wild-type seedlings
immediately after germination but also that the rate of growth was retarded in
the
mutants (Figure 5). In addition, dwf4 hypocotyls reached their terminal length
in <S
days, whereas wild-type seedlings continued to grow.
In sum, the dwf4 phenotype can be described as being due to both primary and
secondary effects of reduced cell elongation. The primary effect is simply a
reduction
in the length of individual organs exclusively along their normal growth axis;
that is,
organ width is not reduced (Table 1). The secondary effects of reduced cell
elongation
are themselves due to the reduction in organ length. The dark green color of
the
leaves, for example, may be due exclusively to the existence of a wild-type
number of
chloroplasts in a significantly smaller cell. Similarly, the sterility of
mutants is a
consequence of the shortness of the stamens, which fail to deposit their
pollen on the
stigmatic surface. In addition to the morphological alterations of dwf4,
mutants
display delayed development, the first sign of which occurs at flowering
(Table 1).
Because rosette leaves are produced continuously during vegetative
development,
delayed flowering results in dwf4 rosettes having almost twice the number of
leaves
observed in the wild type.
Example 5: The Growth Defect of dwf4 Is Due to a Reduction in Cell Length
Both the short stature and the reduced growth rate of dwf4 could be due to a
defect in cell division or cell elongation or both. To distinguish between
these
possibilities, we analyzed sections from 7-day-old hypocotyls and 5-week-old
inflorescence stems, by light microscopy, as described in Example 1. To
minimize
variations due to the developmental stage of the sample, we always took the
stem
sections from the fourth internode. As shown in Table 2, the average cell size
in dwf4
is significantly smaller than in wild-type plants, whereas no differences were
detected
in the number of cells along the length of either organ between the wild type
and
dwf4. Therefore, the short stature and reduced organ length of dwf4 are
largely or
exclusively due to a failure of individual cells to elongate. No differences
were
observed in the number of cell layers contained in the wild type and dwf4.
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Table 2: Cell Length in Wild-Type
and dwf4 plants
Measurement Wild-type dwf4
average cell length in hypocotyl:92.7 qm 32.2 qm
7 day old
plant
average cell length in stem: 79.2 qm 15.0 ~m
5-week old plant
The small size of dwf4 cells offers a possible explanation for the dark green
color of the mutant plants. Chlorophyll measurements were taken, and leaf
mesophyll
protoplasts were prepared, stained, and measured to visualize and count
chloroplasts,
as described in Methods. Although there were no significant differences in
total
chlorophyll content, the chlorophyll a/b ratio, or the absorption spectra
between
wild-type plants and mutants, the mean plane area (the apparent two-
dimensional
surface area of mounted cells) of dwf4 leaf mesophyll protoplasts was 376 mm',
whereas that of wild-type protoplasts was 599 mm2. The two-dimensional
comparison
of plane area represents a dramatic reduction in volume for dwf4 cells.
However, the
number of chloroplasts per cell was only slightly lower: the mean number of
chloroplasts per cell was 40 for dwf4 and 44 for the wild type. Therefore,
dwf4 cells
contain a greatly increased number of chloroplasts per unit cell volume. As a
consequence, the chloroplasts are brought closer to each other, making the
color of the
leaves appear darker. Chloroplast size was the same in both lines.
Thus, the rate of growth was significantly reduced in agar-grown dwf4
seedlings, which ceased to grow when their hypocotyl length was <20% of the
final
wild-type length. Because all of the cells in a hypocotyl before the
initiation of leaf
development are present in the embryo, the initial growth of seedlings is due
exclusively to cell expansion, which therefore must be reduced in dwf4. A
similar
situation applies to soil-grown plants. Five weeks after germination, well
after plants
had bolted, dwf4 plants were shorter than wild-type plants (Table 1). Although
the
mutants continued growing for several weeks more than did the wild type, they
remained shorter through senescence. That cell elongation is the direct cause
of this
decreased growth is shown by measurements of cell length both in 7-day-old
hypocotyls (Table 2) and in 5-week-old stems (Table 2). Not only is the
reduction in
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cell length in good agreement with the reduction in organ length, but insofar
as could
be determined, there is no difference in the number of cells between dwf4 and
wild-type plants.
Organ growth by cell elongation in plants occurs as part of normal
S development in response to a variety of input signals. Mutants that are
defective in
these signaling pathways invariably fail to elongate normally in response to
the
appropriate stimuli. A mutant with a block at a step that is common to several
individual pathways would therefore be expected to have defective responses to
all of
the corresponding signals. dwf4 appears to be such a mutant. Figure 6 shows
that
elongation induced by the hy2 mutation is blocked in a dwf4 hy2 double mutant.
Not
surprisingly, in view of this result, dwf4 also failed to display hypocotyl
elongation as
a response to growth in complete darkness. In addition, dwf4 was capable of
perceiving GA, but its response was severely compromised. This mutant could
also
respond to the inhibitory effects of auxin but was incapable of auxin-
stimulated
elongation. It was only exogenous BL that fully restored wild-type length to
dwf4
hypocotyls (Choe et al. (1998), supra).
Because dwf4 failed to respond to at least three independent signaling
pathways but responded fully to only one, the most likely explanation for the
dwarf
phenotype is therefore that a fully functional BR system is required for a
full response
to GA, auxin, and deetiolation. From the perspective of cellular economy, it
may be
advantageous that the downstream elements involved in cell elongation are
shared
among at least some of the signaling pathways that evoke this response. The
interaction of various pathways at a common step provides the plant with a
potential
point for the integration of signals produced by diverse independent stimuli.
Our
results indicate that BRs act at this downstream step.
Example 6: dwf4 Is Specifically Rescued by Brs
The reduced length of cells in dwf4 hypocotyls and inflorescence stems is
indicative of a failure of these cells to elongate during development. A
variety of
endogenous and environmental signals is responsible for stimulating elongation
in
plants; therefore, a series of experiments was performed to determine whether
dwf4 is
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affected in a specific signaling pathway or is blocked in elongation as a
response to
various signals.
Of the endogenous (hormonal) signals that might be deficient in dwarf plants,
an obvious candidate is GA, because gibberellin-deficient mutants are shorter
in
stature than are the wild-type plants (Koornneef and Van der Veen, supra). Our
results, however, indicate that dwf4 is not defective in the synthesis of
gibberellins.
When germinated on 10-5 M GA, wild-type seedlings demonstrated an elongation
response (Figure 6), whereas dwf4 seedlings responded minimally, if at all. At
10-4 M
GA, wild-type seedlings elongated slightly more than at 10-5 M, but the dwf4
seedlings were essentially saturated for elongation at 10-5 M GA. Similar
results were
obtained when soil-grown plants were sprayed with 1 mM GA once inflorescences
first became visible: dwf4 inflorescence stems elongated by only 28% above the
untreated controls, whereas those of the wild type elongated by 45% above the
untreated controls. Mutants that owe their reduced stature to decreased levels
of
endogenous gibberellins can be fully rescued by added hormone (Koornneef and
Van
der Veen, supra; Talon et al., supra). In addition, dwf4 seeds germinate in
the absence
of exogenously supplied GA. Our results therefore suggest that dwf4 is not
deficient
in endogenous GA. A corollary conclusion from this experiment is the
demonstration
that dwf4 is capable of detecting GA; that is, it is not likely to be affected
in signal
perception but rather is defective in the extent to which it can respond to
this signal.
Auxin can also stimulate cell elongation. This effect is especially visible in
young seedlings (Klee and Estelle, supra). The response of wild-type and dwf4
plants
to auxin was tested by growing seedlings for 10 days on vertically oriented
plates
containing various concentrations of the synthetic auxin 2,4-D. At all
concentrations
assayed, inhibition of root growth was evident. Figure 6 shows that at 10-8 M
2,4-D,
hypocotyl elongation in wild-type and dwf4 seedlings was similar to that of
the
controls. Higher concentrations of auxin were inhibitory for both wild-type
and dwf4
seedlings, and lower concentrations had no effect. In view of the inhibition
of root
growth, it is clear that dwf4 is not auxin resistant; rather, its elongation
response is
compromised.
As mentioned above, the most obvious exogenous signal for plants is light.
Therefore, to investigate whether light-regulated cell elongation is altered
in dwf4,
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wild-type and dwf4 seedlings were grown in the dark, as described in Example
1.
Figure 6 shows that as expected, wild-type seedlings displayed hypocotyl
elongation
typical of etiolated growth. By contrast, dark-grown dwf4 seedlings were only
slightly longer than those grown in the light. To assess the relationship
between the
dwf4 phenotype and light sensing by dwf4, the mutation was crossed into a
mutant
defective in the HY2 gene. All by mutants share the common phenotype of an
elongated hypocotyl that mimics part of the etiolation response in the light.
Specifically, hy2 is deficient in active phytochrome because chromophore
biosynthesis does not take place (Chory et al. (1989a) Plant Cell 1:867-880).
Figure 6
shows that dwf4 hy2 double mutants displayed a dwarfed phenotype
indistinguishable
from that of dwf4 HY2 (light-grown control); therefore, the elongation block
due to
the dwf4 mutation is epistatic to a defect in phytochrome activity.
In the course of our studies, we prepared a genomic library from dwf4-l, from
which we isolated a clone in which a fragment of T-DNA interrupts a gene
encoding a
putative cytochrome P450 steroid hydroxylase. Because BRs have been shown to
elicit elongation in Arabidopsis (Clouse et al. (1993) J. Plant Growth Regul.
12:61-66) and because BR-deficient mutants have been recently described
(Kauschmann et al. (1996), supra; Li et al. (1996), supra; Szekeres et al.,
supra ), we
tested the effect of BL on Arabidopsis seedlings by germinating seeds in
liquid
medium containing different amounts of BL. As shown in Figure 6, the dwf4
hypocotyls were restored to wild-type height by 10-6 M BL. This, together with
our
identification of a disrupted gene encoding a putative BR biosynthetic enzyme,
strongly suggests that the phenotype of dwf4 is specifically due to a defect
in BR
biosynthesis (see Choe et al. (1998) Plant Cell 10:231-243).
Thus, the results indicate that BL is involved at or near a downstream control
point where multiple signaling pathways interact. First, as shown in Figure 6,
BL is
required for cell elongation as a response to darkness as well as GA and
auxin. In
addition, previous studies (Kauschmann et al. ( 1996), supra; Li et al. (
1996), supra;
Szekeres et al. ( 1996), supra) and the work described herein show that BR can
compensate for the cell elongation defect of mutants as diverse as det2, cpd,
dwf4,
detl, copl, and dwfl. This places BRs downstream of all the cellular functions
affected in these mutants. Finally, at least one of the BR biosynthetic genes
has been
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shown to be modulated by light, cytokinins, and the carbon source (Szekeres et
al.
(1996), supra).
Mutations in axr2 result in a dwarf growth habit and a dark-grown phenotype
with short hypocotyl and open cotyledons (Timpte et al. (1992), supra). In
addition,
axr2 mutants are resistant to auxin, ethylene, and abscisic acid and have
defective root
and shoot gravitropism. The dwarf phenotype in axr2 mutants has been shown to
be
due to reduced cell elongation and is rescued by BL (Szekeres et al. (1996),
supra).
This suggests that at least one of the multiple hormone signaling pathways
affected in
axr2 involves a BR-dependent step. Mutations at another locus, acaulisl, also
have a
significant reduction in cell elongation, but the defect is confined to
inflorescence
stems and leaves (Tsukaya et al. (1993) Development 118:751-764). Flowers are
fully
fertile and mature into normal-sized siliques with normal seed set. There is
no change
in hypocotyl length. If BRs are directly involved in this apparently organ-
specific
signaling pathway, it may be due to organ-specific responsiveness to
individual BR
species. With regard to the mechanism of action of BRs, at the moment one can
only
speculate that the target may be a component of the cell expansion machinery.
Perhaps steroid signaling initiates a series of events leading to cell wall
loosening.
Example 7: The Elongation Defect of dwf4 Leads to a Light-Regulatory
Phenotype
The BR-deficient mutant det2 was originally identified as defective in
regulation by light (Chory et al. (1991), supra). Given the similarity of det2
and dwf4
phenotypes and functions and in view of the observation that dwf4 is epistatic
to hy2,
one can predict that the etiolation response, which includes significant
hypocotyl
elongation, would not be normal in dwf4. To assess to what extent the
etiolation
response is affected by BR-dependent cell elongation, we grew dwf4 and wild-
type
plants on agar under continuous light or in complete darkness, as described
above in
Example 1. After 7 days of growth in the light, wild-type seedlings displayed
open
and expanded cotyledons as well as emerging leaf buds. In contrast, the
overall
appearance of light-grown dwf4 seedlings was strikingly similar to that of
det2 (Chory
et al. (1991), supra). dwf4 hypocotyls were very short, and the cotyledons
were
smaller than those of the wild type, displaying significant epinastic growth.
As
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expected, dark-grown wild-type seedlings had a typical etiolated appearance,
with a
highly elongated hypocotyl and closed, unexpanded cotyledons. However, dwf4
hypocotyls failed to elongate. That the dwf4 mutation can abolish the
elongation
component of the etiolation response is in agreement with the notion that the
block in
cell elongation in dwf4 is specifically a BR-dependent process.
In addition to short hypocotyls, dark-grown dwf4 seedlings displayed partially
open cotyledons and leaf primordia, with up to four leaf buds clearly visible.
This has
not been observed with the wild type, although it occurs with certain light-
regulatory
mutants (Chory et al. (1989b), supra; Deng et al. (1991) Genes Dev. 5:1172-
1182;
Wei and Deng (1992), supra). dwf4 leaf development continued in the darkness
for
several weeks, resulting in significant expansion of rosette leaves. These
results
indicate that dwf4 plants can initiate what is normally a photomorphogenic
pathway in
the absence of light. Although this is often diagnostic of a light-regulatory
mutant,
wild-type Arabidopsis can perform leaf development and even flowering in
complete
darkness when grown in liquid culture (Araki and Komeda (1993) Plant J.
4:801-811).
The cause for this dark-flowering effect is not understood; therefore, the
possibility exists that leaf development in dark-grown dwf4 is related to dark
flowering and not to a light-regulatory defect. For example, perhaps the
proximity of
the dwf4 shoot apical meristem to the surface of the agar, due to the
shortness of the
hypocotyls, mimics some effect of submerged culture, such as a high water
potential
or a high concentration of some nutrient. To test this possibility, wild-type
seedlings
were grown in complete darkness for 6 weeks in vertically oriented dishes to
maximize contact between the seedling and the medium. Wild-type seedlings
grown
in this fashion displayed open cotyledons and underwent at least partial leaf
development. In fact, all wild-type seedlings grown along the surface of the
agar
showed development of an inflorescence with at least one cauline leaf and a
terminal
flower bud. We conclude, therefore, that the appearance of leaves in dark-
grown dwf4
may be due simply to its short size and the culture conditions.
A number of light-regulatory mutants have been described that undergo
photomorphogenesis in the dark at the cellular level. In mutants such as copl,
cop8,
cop9, copl0, and copll, stomata undergo photomorphogenic maturation (Deng and
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Quail (1992), supra ; Wei and Deng (1992), supra ; Wei et al. (1994), supra );
of
these, copl and cop9 as well as detl (Chory et al. (1989b), supra) also
initiate
differentiation of plastids into chloroplasts. To determine whether dwf4
plants
undergo photomorphogenic cellular differentiation in the dark, we analyzed
cotyledons from light- and dark-grown plants by transmission and scanning
electron
microscopy. Analysis of plastids in thin sections from 7-day-old dark-grown
seedlings showed no difference between the wild type and dwf4. Both lines
contained
normal chloroplasts when grown in the light, whereas dark-grown seedlings
contained
etioplasts, with their characteristic prolamellar body and no significant
organization of
thylakoids. Analysis of stomatal structures on the underside of cotyledons
from
7-day-old seedlings indicates that stomatal development was not completed in
the
dark, because the stomatal opening was occluded in both lines. The majority of
light-regulatory mutants analyzed to date displayed light-grown morphology in
the
dark without concomitant chloroplast or stomatal development. As in these
mutants,
therefore, the dwf4 mutation uncouples the developmental pathway of seedling
morphology from that of light-regulated cellular differentiation.
An additional feature of many light-regulatory mutants is that
photomorphogenesis in the dark is accompanied by expression of genes that
normally
are light induced (Chory et al. (1989b), supra , Chory et al. (1991), supra ;
Deng et al.
(1991), supra ; Wei and Deng (1992), supra ; Hou et al. (1993), supra ; Wei et
al.
( 1994), supra ). To assess whether dwf4 is able to induce light-regulated
transcripts in
the dark, we compared the activity of a CAB promoter fused to the Escherichia
coli
gene uidA, encoding ~i-glucuronidase (GUS), in light- and dark-grown dwarf and
wild-type plants. The CAB-uidA fusion in pOCA107-2 (Li et al. (1994) Genes
Dev.
8:339-349 ) was crossed into dwf4, and F2 dwarf and wild-type plants were
grown in
the dark or light for 12 days, followed by determination of GUS activity by
fluorometry (Gallagher (1992). "Quantitation of GUS activity by fluorometry"
in
GUS Protocols, S.R. Gallagher, ed (New York: Academic Press), pp. 47-59).
The results demonstrated that when grown in the light, both wild-type and
dwf4 seedlings contained GUS activity, which was significantly reduced in both
lines
when grown in the dark. Moreover, dark-grown dwf4 seedlings displayed no GUS
activity above the background present in dark-grown wild-type plants. The
absence of
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light-induced gene expression in the dark is a distinguishing feature of
certain cop and
det mutants, such as cop2, cop3, and det3. Because we have shown that the
defect in
cell elongation of dwf4 is specifically rescued by BRs, even in the presence
of light,
we conclude that this is not a light-regulatory mutant. That its phenotype is
partially
deetiolated or constitutively photomorphogenic is a secondary effect of its
reduced
stature and the growth conditions.
Example 8: Abnormal Skotomorphogenesis as a Consequence of the Dwarf
Growth Habitat
When dwf4 is grown in the light, its morphology is similar to that of various
cop and det mutants, with multiple short stems, short rounded leaves, loss of
fertility
due to reduced stamen length, and delayed development (Figure 6). Dark-grown
dwf4
seedlings possess short hypocotyls, open cotyledons, and developing leaves.
Therefore, it is tempting to speculate that this mutant may be defective in
the control
1 S of light-regulated processes. On the other hand, because a dark-flowering
phenotype
has been demonstrated for liquid-grown Arabidopsis (Araki and Komeda (1993),
supra), and given that agar medium is mostly water, it is especially
significant that it
is the dwarf seedlings, whose apical meristems are very close to the agar
surface, that
display a light-grown phenotype in the dark. Furthermore, because wild-type
seedlings grown along the surface of the agar reproduce the dark-flowering
phenotype, it is possible that the apparent light-regulatory defect of dwarf
seedlings is
a dark-flowering response. This possibility is strengthened by the observation
that
wild-type seedlings (ecotype Wassilewskija [Ws-2]) grown in the dark on
horizontally
oriented plates occasionally bend down and touch the agar surface, and these
seedlings invariably produce leaves.
In addition, of the eight DWF loci identified in this laboratory, only the
shortest mutants displayed open cotyledons and leaf bud development; in the
case of
dwfl (Feldmann et al. (1989), supra), this aberrant skotomorphogenesis is
confined to
the most severely affected alleles. In addition to the presence of a short
hypocotyl and
at least partially open cotyledons in the dark, copl (Deng and Quail (1992),
supra),
detl (Chory et al. (1989b), supra), and det3 (Cabrera y Poch et al. (1993)
Plant J.
4:671-682) have been shown to initiate leaf formation in the dark. In mutants
such as
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copl, cop8, cop9, copl0, and copll, stomata undergo photomorphogenic
maturation
(Deng and Quail (1992), supra; Wei and Deng (1992), supra; Wei et al. (1994)),
supra); of these, copl and cop9 as well as detl (Chory et al. (1989b), supra)
also
initiate differentiation of plastids into chloroplasts. dwf4 displayed, in
addition to a
light-grown dwarf phenotype, a dark-growth phenotype of short hypocotyls, open
cotyledons, and developing leaves; however, in contrast with the light-
regulatory
defect seen with whole plants, the cellular differentiation phenotype was
unaffected.
In dark-grown dwarf seedlings, stomata did not complete their development, and
differentiation of chloroplasts was not observed. The absence of a cellular
light-regulatory phenotype in dwf4 is similar to that of a number of
photomorphogenic mutants, such as det2, det3, cop2, cop3, and cop4 (Chory et
al.
(1991), supra; Cabrera y Poch et al. (1993), supra; Hou et al. (1993), supra).
In view of the dark-flowering phenotype on agar and the absence of a
light-regulatory defect in differentiating cells, we conclude that at least in
the case of
dwf4, aberrant skotomorphogenesis may be a consequence of a dwarf growth habit
rather than dwarfism being part of a defect in the control of light-regulated
processes.
This effect may also explain the light-regulatory phenotype found in other
mutants
with severely reduced height, such as axr2 (Timpte et al. (1992), supra), and
strong
alleles of dwfl , both of which are also rescued by exogenous BRs (Szekeres et
al.
( 1996), supra).
Example 9: Feeding Experiments with BR Biosynthetic Intermediates
In view of the results described above, we hypothesized that DWF4 mediates
one or more of several steroid hydroxylation steps in the BR biosynthetic
pathway.
To test this, dwf4 was grown on all of the available biosynthetic
intermediates in the
BR biosynthetic pathways and examined to ascertain which intermediates could
rescue the dwarf phenotype. In addition to the intermediates belonging to the
early
C-6 oxidation and late C-6 oxidation pathways (Choi et al. (1997), supra),
22a-hydroxycampesterol (22-OHCR), 6a-hydroxycathasterone (6-OHCT) (Takatsuto
et al. (1997) J. Chem. Res. (synop.) 11:418-419), and 6a-hydroxycastasterone
(6-OHCS) (S. Takatsuto, T. Watanabe, T. Noguchi, and S. Fujioka, unpublished
data)
were synthesized and tested.
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Germinated seedlings were transferred to media supplemented with one of the
intermediates or BL to pinpoint the step catalyzed by DWF4. Cathasterone (CT;
early
C-6 oxidation pathway), 6-OHCT, 6-deoxocathasterone (6-deoxoCT; late C-6
oxidation pathway), and 22-OHCR, and all of the downstream compounds belonging
to each branch, rescued the light-grown dwf4 phenotype, whereas the known
precursors failed to cause an elongation response. Rescued seedlings exhibited
greatly elongated cotyledonary petioles and expanded cotyledons, moderately
elongated hypocotyls, and leaves that were larger and not as curled compared
with
nonrescued dwarfs. In addition, the rescued seedlings were less green than the
dwarfs. These experiments were conducted in liquid media. Feeding experiments
performed in the dark yielded similar results.
Dose-response tests on the putative substrates and products of DWF4 were
also performed. dwf4 seedlings failed to respond to 6-oxocampestanol (6-oxoCN)
even at high concentrations (3 x 10-6 M). However, on CT the overall
morphology of
1 S dwf4 was essentially rescued to wild-type phenotype at 3 x 10-' M and
higher,
whereas with 6-deoxoCT, rescue occurred with as little as 10-' M and may have
even
been inhibitory at higher concentrations. Of particular interest is the more
dramatic
response of the epicotyls versus the smaller response of the hypocotyls to CT.
This
same phenomenon was true for seedlings treated with >10-' M 6-deoxoCT. At
concentrations >10-' M, the seedlings displayed an inhibition in hypocotyl and
root
elongation as well as cotyledon and leaf expansion.
In a dose-response experiment performed in the dark, the seedlings failed to
respond to 6-oxoCN (10-8 to 3 x 10-6 M). A higher concentration of CT for
dark-grown seedlings, compared with light-grown seedlings, 3 x 10-6 M (Figure
SB),
was required to convert the hypocotyl to a length similar to that of the wild
type.
High concentrations of 6-deoxoCT caused dramatic elongation but were less
effective
at rescuing dwf4 hypocotyls to wild-type phenotype.
To determine whether the results of the seedling feeding experiments could be
applicable to soil-grown mature plants, 6-week-old dwf4 plants were treated
with BR
intermediates and BL. Concentrations of applied intermediates were adjusted
empirically to optimize responses. Consistent with the results obtained from
the
seedling experiments, only 22a-hydroxylated compounds can rescue the dwf4
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phenotype. The elongation response was only observed in the young tissues of
the
inflorescence, regardless of whether the BRs were applied locally or sprayed
over the
entire plant. In contrast to the striking elongation of the peduncles and
pedicels,
fertility was not restored by BR treatment. The sterility in dwf4 is
hypothesized to be
mechanical, which means that the filaments are shorter than the carpels such
that the
pollen is shed onto the ovary walls rather than onto the stigmatic surface. In
fact, if
dwf4 plants are hand pollinated using dwf4 pollen, fertility increases.
Pedicels displayed a more consistent response to exogenously applied BRs
than did internodes, which led us to quantify the sensitivity of pedicels to
these
compounds. As shown in Figure 7, dwf4 pedicels were more sensitive to BR
intermediates belonging to the late C-6 oxidation pathway, 6-deoxoCT (10-6 M)
and
6-deoxoteasterone (6-deoxoTE; 106 M), compared with CT (10~' M) and teasterone
(TE; 10-5 M) of the early C-6 oxidation pathway. The end product of the BR
pathway,
BL (10-' M), possessed the highest bioactivity. This concentration induced
1 S approximately the same degree of response as its precursor compounds at 10-
6 M.
Finally, application of 22-OHCR (10-5 M) also resulted in a dramatic
elongation
response (Figure 7).
Rescue of dwf4 by 22-hydroxylated steroids confirms that the missing step in
dwf4 is hydroxylation at the C-22 position. In fact, we found that the
chemically
synthesized 22-OHCR was also effective in rescuing dwf4 (Figure 7)
hydroxylation at
C-22. These results indicate that there is no defect other than 22a-
hydroxylation in
dwf4 plants.
In BR biosynthesis, Fujioka and Sakurai (1997b), supra have demonstrated
that there are at least two branched biochemical pathways to the end product
BL
(Figure l; Fujioka and Sakurai (1997a), supra, Fujioka and Sakurai (1997b),
supra;
Sakurai and Fujioka (1997), supra). Depending on the oxidation state of C-6,
they are
referred to as the early or late C-6 oxidation pathways. In the early pathway,
the C-6
is oxidized to a ketone at campestanol (CIA, whereas in the late pathway it is
oxidized
at 6-deoxocastasterone (6-deoxoCS). Otherwise, the two pathways share
equivalent
reactions. Our results from the experiments with the available BR
intermediates
clearly demonstrate that dwf4 is defective in the 22a-hydroxylation steps in
each of
the pathways. Application of all 22a-hydroxylated intermediates in these
pathways,
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such as CT and 6-deoxoCT, cause dramatic elongation of dwf4 plants, but
compounds
not hydroxylated at C-22 had no effect. This result also suggests that DWF4
recognizes at least two substrates: CN and 6-oxoCN. It seems reasonable to
hypothesize that the same result will be found for CPD, a 23a-hydroxylase;
that is, it
will use 6-deoxoCT as well as CT as substrate.
The rescue of dwf4 by 22-OHCR is an important observation. First, it
confirms DWF4 as a 22a-hydroxylase. Second, this result suggests that 22-OHCR
was metabolized to induce the same responses as other complementing BRs. This
is
not just a general effect because our unpublished data show that another dwarf
mutant
that we have identified in our screens, dwf$-1, is not rescued by this
compound.
Finally, these feeding experiments suggest that the metabolism of 22-OHCR may
represent a new subpathway in the BR biosynthetic pathway. If this compound
also
exists in vivo and constitutes the first step in a separate subpathway, by
analogy to the
chemical structure, the C-6 hydroxylated BRs, for example, 6-OHCT,
6-hydroxyteasterone, and so on, may be possible intermediates in this network.
If so,
the intermediates in this pathway may play a role as bridging molecules
between the
early and late C-6 oxidation pathways. Alternatively, it might be possible
that
22-OHCR merges into one of the two pathways to be metabolized. In this case,
the
late C-6 oxidation pathway is the best candidate; our unpublished data show
that
22-OHCR is more effective in the light in rescuing the dwf4 phenotype, which
is true
for all of the intermediates in the late C-6 oxidation pathway.
Currently, biochemical feeding studies suggest that the two pathways merge to
produce BL or CS (Yokota et al. (1991), Metabolism and biosynthesis of
brassinosteroids. In Brassinosteroids: Chemistry, Bioactivity, and
Application, H.G.
Cutler, T. Yokota, and G. Adam, eds (Washington, DC: American Chemical
Society),
pp. 86-96; Yokota, et al. (1997) Plant Physiol. 115(suppl.):169; Figure 1).
Several
lines of evidence indicate that seemingly redundant pathways can be utilized
to
respond to environmental or developmental signals. First, the pathways could
respond to specific signals. For instance, it is possible that various cues
such as light,
dark, or developmental signals play a role in regulating these subpathways.
Our
feeding experiments consistently showed that BRs in the late C-6 oxidation
pathway
are more effective at promoting cell elongation in light-grown plants (dwf4
and wild
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type; Figure 7) and that the BRs belonging to the early C-6 oxidation pathway
are
more active in dark-grown seedlings. Thus, it may be possible that the late C-
6
oxidation pathway operates in the light and that the early C-6 oxidation
pathway
functions primarily in the dark. Second, rather than a simple merger of
branched
pathways to BL as an end product, each intermediate may have nascent
bioactivity.
The in vivo ratio or composition of BRs at different oxidation states may
result in
different responses. Noticeably distinctive phenotypes for the various BR
dwarfs,
defective in different biosynthetic steps, support this idea. Third, the
biosynthetic rate
of each pathway toward production of the end product may differ. In this case,
the
biosynthetic rate could be modulated by controlling the level of gene
expression or
the activity of participating enzymes. Certain signals, requiring different
rates of BR
biosynthesis, may induce one of the subpathways, which would then affect the
concentration of the intermediates in one pathway relative to the other.
Of the steps in BR biosynthesis in Madagascar periwinkle, the
22a-hydroxylation reaction has been suggested to be the rate-limiting step
(Fujioka et
al. (1995a) Biosci. Biotech. Biochem. 59:1543-1547). In periwinkle, the
endogenous
level of CT was as low as one-twenty thousandth of CR; however, CT was almost
500
times more active than 6-oxoCN in the rice-lamina inclination assay (Fujioka
et al.
(1995b) Biosci. Biotech. Biochem. 59:1973-1975). Based on these results, we
propose
that the step encoded by DWF4 serves as the rate-limiting reaction and that
once past
this step, the intermediates are easily converted to the end product. Although
biochemical studies on DWF4 need to be performed to ascertain whether it
mediates
the rate-limiting step, DWF4 seems to be greatly downregulated compared with
CPD,
the next enzyme in the pathway; RT-PCR revealed that the DWF4 transcript is
much
less abundant than the CPD transcript.
Example 11:
A. Promoter and overexpression constructs
Two promoter constructs were used for the DWF4 promoter:: GUS (D4G)
analysis. For promoter fusions, polymerise chain reaction (PCR) products
spanning
1.1 kb DNA upstream of the translation initiation site were amplified using
primers
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D4XLIT1IT (5'-TAGGATCCAGCTAGTTTCTCTCTCTCTCT-3') (SEQ ID N0:16)
and a T7 primer (5'-TAATACGACTCACTATAGGG-3') (SEQ >D N0:17). For
template for PCR, a DWF4 genomic clone subcloned into pBluescript SK- vector
(Stratagene, La Jolla, CA) was used as described herein. The PCR products were
S restricted with SaII and BamHI, and ligated into the same restriction site
of a
promoterless GUS vector pBI101; this 1.1 kb promoter:: GUS construct was named
pD4GL. For the pD4GS construct, pD4GL was digested with HindIII, the small
restriction fragment was removed, and the remaining vector with the partial
promoter
was self ligated. The constructs were introduced into Agrobacterium strain
GV3101
through electroporation.
For a DWF4 overexpression construct, PCR products were made by using
D40VERFA (5'-GAATTCTAGAATGTTCGAAACAGAGCATCATA-3') (SEQ ID
N0:18) and D4R2 (5'-CCGAACATCTTTGAGTGCTT-3') (SEQ ID NO:10) primers
and Wassilewskija-2 (Ws-2) genomic DNA. The PCR products were cut with XbaI
and HindIII, and inserted into the same restriction sites of genomic clone
SCH25
containing a 2.5 kb HindIII fragment of the DWF4 DNA corresponding to the 3'
half
of the gene. The resulting recombinant DNA clone pD4CDS, containing the whole
coding sequence from the translation initiation site to 694 by downstream of
the stop
codon, was cut with XbaI and transferred to an overexpression vector pART27
(Gleave (1992), Plant Molec. Bio. 20:1203-1207). The resulting binary
construct was
named pOD4. This construct was introduced into Agrobacterium through
electroporation.
B. Spray transformation
Since it has been shown that Agrobacterium-mediated transformation can
work by seed infection (Feldmann and Masks (1987) Molec. Gen. Genet. 208:1-9)
or
by simply dipping the host plants into Agrobacterium culture, we decided to
try
spraying the Agrobacterium directly onto the plants. In addition to spraying ,
the
"floral dip" method was used as described (Clough and Bent (1998), infra).
About 20
Wassilewskija-2 (Ws-2) wild-type and dwf4-4 seeds were sprinkled on 10 cm
pots,
and thinned to 5-6 plants per pot 10 days (wild type) and 20 days (dwf4-4)
after
germination. When the primary inflorescences of the wild type reached 3-4 cm
in
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height, they were decapitated to induce axilary bolts. dwf4-4 plants were used
without
decapitation. For the preparation of Agrobacterium, a single colony selected
on 20
~g/ml kanamycin in Luria-Bertani (LB) medium (10 g bacto-tryptone, 5 g bacto-
yeast
extract, 10 g NaCI per liter, pH 7) was inoculated into 100 ml liquid LB
media, and
grown for 3 days. One ODboo unit equivalent cells were used to inoculate 100
ml LB
media. The overnight grown cells were collected by centrifugation, and
resuspended
with transformation media as described in Clough and Bent (1998), infra (5%
sucrose
and 0.05% Silwet L-77, ODboo - 1). The Agrobacterium suspension was sprayed
onto
plants on the third day after decapitation. To avoid physical contact with
possibly
hazardous Silwet vapor, protective glasses were used and the spraying was done
in a
fume hood. To test the efficiency of repeated spraying, plants were sprayed
every
third day (3x). Sprayed plants were grown to maturity and seeds harvested. For
seed
sterilization 0.07 g seeds were surface sterilized by treating for 2 min in
70% ethanol,
min in bleach solution consisting of S% Clorox and 1% SDS, followed by three
1 S rinses with sterile water. To plate the seeds 25 ml of sterile top agar
(0.1 S% agar) was
added to the sterilized seeds and the seed mixture was poured onto Murashige
and
Skoog solid plate (100 x 15 mm, Murashige and Skoog salts, 5% sucrose, 0.08%
agar,
pH 6) supplemented with kanamycin or hygromycin at 60 ~g/ml and 40 pg/ml,
respectively. Twelve days after germination kanamycin resistant were
transferred to
single pots, and grown to maturity. T2 seeds were collected from individual
transformants (T 1 ), and plated again on the selection media to determine
segregation
ratios for drug-resistant versus sensitive plants. Arabidopsis transformants
were
named Arabidopsis Overexpressor of DWF4 (AOD4) when harboring an
overexpression construct pOD4, and DWF4 promoter:: GUS (D4G) for transformants
containing a GUS fusion gene. Homozygosity for the transgene was determined
when
no sensitive T4 seedlings segregated from >500 T3 individuals. Morphometric
analysis of AOD4 lines and GUS histochemical analysis of D4GL plants was
performed using plants homozygous for the transgene.
For histochemical analysis of the D4GL plants, seeds were plated on M&S
plates and grown in the dark and light. Seedlings were harvested at the
designated
dates and stained overnight using a substrate mixture (0.1 M NaP04, pH 7, 10
mM
EDTA, 0.5 mM K3Fe(CN)6, 0.5 mM KQFe(CN)6, 1 mM X-glucuronide, and 0.1
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Triton X-100). Seedlings cleared with 90% ethanol were rehydrated before
taking
pictures using a Stemi SV 11 dissecting microscope (Zeiss, NY).
Transgenic tobacco plants (TOD4) harboring the pOD4 constructs were
produced in the plant tissue culture laboratory at the University of Arizona.
Protocols
for the regeneration of transgenic plants from lead discs of Nicotiana tabacum
var
Samsun will be provided on request. Fifteen independent transformants for both
the
control and OD4 constructs were grown for seeds. Morphological analysis of the
TOD4 lines was performed using T2 plants in the course of growth for 4 months
in
the green house (30°C). Methods for Arabidopsis growth and RNA gel blot
analysis
were previously described herein. Briefly, seeds of wild type and the two AOD4
lines
were germinated on M&S agar media. 10 days after germination, 20 seedlings
confirmed to be resistant to kanamycin were transferred to a single pot.
Various
morphological traits (Table 3) were measured. To determine the seed
production,
after 8 weeks from germination, plants were further dried for two weeks at
room
1 S temperature. Seeds were harvested from an individual plant and weighed. To
measure the seed size, seeds were magnified 3 times under the dissecting
microscope,
the width and the length of five seeds from each plant were measured to the
nearest
tenth of mm.
C. DWF4 transcription is localized to zones of cell division and elongation
To localize BR biosynthesis, RNA gel blot analysis with total RNA isolated
from nine different tissues of three-week old plants was performed. The DWF4
transcript was barely detectable in shoot tips, roots, dark-grown seedlings,
callus and
axilary buds, but the levels were below the detectable limit in the other
tissues
examined, including stems, siliques, pedicels, and rosette leaves. For finer
localization
of the expression, the expression of the GUS reporter gene controlled by the
DWF4
promoter was examined.
Prior to performing DWF4 promoter::GUS gene fusion analysis, a 1.1 kb
fragment of DNA upstream of the DWF4 translation start site was tested to
ensure that
it contained all of the necessary sequence elements for proper transcriptional
control
of DWF4. dwf4-4 plants were transformed with a 4.8 kb construct consisting of
a 1.1
kb promoter region and 3.7 kb that contained the complete DWF4 coding
sequence.
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For Agrobacterium-mediated transformation of Arabidopsis plants, a "spray
transformation" protocol was employed rather than traditional methods. Spray
transformation yielded a comparable number of transformants relative to the
traditional "floral dip" (Clough and Bent (1998) Plant J. 16:735-743) or
"vacuum
S infiltration" methods (Bechtold et al. (1998) Methods Mol. Biol. 82:259-
266).
Interestingly, repeated spraying resulted in an increased number of
transformants.
Transformants harboring the DWF4 genomic DNA displayed a wild type phenotype,
suggesting that the promoter segment contained the necessary information for
proper
expression of the gene.
For histochemical staining analyses of transgenic plants harboring the DWF4-
promoter.: GUS (D4G) recombinant gene, two different D4G constructs were made
and tested. D4GL contained the 1.1 kb promoter fragment, whereas D4GS carned
only a TATA-like promoter region (280 bp). GUS staining in 20 independent
transformants containing D4GS was either not detected or inconsistent between
transformants. However, the 20 transgenic plants containing D4GL displayed a
consistent GUS staining pattern, suggesting that the 1.1 kb promoter is
required for
the proper transcriptional control of DWF4.
Analyses of GUS staining patterns in T2 plants homozygous for D4GL
revealed that GUS activity was present in tissues with actively dividing or
elongating
cells. These include shoot apical meristems, leaf primordia, collet (the
junction
between hypocotyl and root), and root tips, including lateral root primordia,
as shown
in 6-day old light-grown seedlings. Interestingly, dark-grown seedlings
displayed
GUS activity in cotyledons whereas the staining was not detectable in the
cotyledons
of light-grown seedlings. In adult plants, GUS activity was detected in floral
primordia, carpets, and the basal end of the filaments of unopened flowers,
whereas
GUS activity in sepals, petals, and mature pedicels was not detected. The
shoot tips,
bases of emerging branches, and primordia of axilary inflorescences were GUS
positive, whereas elongated internodes were negative. Embryos in the seeds of
the
fully elongated siliques were weakly positive for GUS staining, suggesting a
role for
BRs in embryo development. Leaf primordia, young leaves, expanding leaf
margins,
and the base of petioles displayed GUS activity, but old leaf blades were
negative for
GUS staining. The tissues positive for GUS staining confirmed the expression
pattern
CA 02362603 2001-08-10
WO 00/47715 PCT/US00/03820
examined by northern analysis with the tissue-specific RNA.
Since DWF4 is proposed to be a key enzyme in the BR biosynthetic pathway,
DWF4 transcription could be regulated by an end-product feedback mechanism. To
this end, D4GL was expressed in different genetic backgrounds including two BR
deficient mutants, dwf7-l and dwJ$-1, and a BR-enriched line, AOD4. GUS
activity
was increased in dwf7-1 and dwf8-1 but decreased in AOD4 lines. DWF7 is a C-5
desaturase that acts in the sterol specific part of the pathway. D4GL activity
in dwf7-1
was found in the same tissues as wild type but. dwf8-1 is defective in a BR
biosynthetic step downstream of CPD. In dwJB-l, the intensity of the D4GL
activity
was noticeably stronger as compared to wild type but the expression patterns
were
relatively diffuse. dwf8-I was also found to express GUS at nascent sites as
compared
to wild type. In wild type, D4GL expression in the cotyledons of light-grown
seedlings was not detected, but dwf$-1 displayed considerable D4GL activity in
the
cotyledons. Also in contrast to wild type, GUS activity was detected
throughout the
hypocotyls of dwfg-1 light-grown seedlings, suggesting that D4GL transcription
is
upregulated in dwf8-1 in a more general manner. Conversely, GUS activity was
greatly reduced in AOD4-4 plants. Also, in AOD4 plants GUS activity in the
root tip
and collet was completely eliminated, whereas the shoot tip retained residual
activity,
suggesting that increased levels of BRs in AOD4-4 may have resulted in lower
GUS
activity. The down-regulation of GUS activity was similarly found if D4GL
plants
were exogenously supplied with 10-6 M 24-epibrassinolide (epi-BL). Seedlings
treated with epi-BL displayed greatly reduced GUS activity in tissues normally
stained in untreated control plants, suggesting that exogenously applied epi-
BL
effectively down-regulates D4GL activity. However, hypocotyls of D4GL plants
supplemented with 10~ M GA3, while longer than controls, did not display an
increase in GUS staining in shoots and roots. This suggests that GA3 or GA3-
induced
elongation did not affect D4GL transcription in these tissues.
D. DWF4 overexpression results in elongated hypocotyls in Arabidopsis and
tobacco seedlings
A DWF4 overexpression construct (pOD4) was made by placing the DWF4
genomic DNA under the control of the CaMV 35S promoter. RNA gel blot analysis,
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WO 00/47715 PCT/US00/03820
with total RNA isolated from the transgenic lines containing the
overexpression
construct, showed that DWF4 transcripts were greatly increased in both
Arabidopsis
and tobacco, whereas the level was not readily detectable in either wild type
or in
dwf4-1 plants. Similar to increased mRNA transcripts, the 80 independent AOD4
transgenic plants had longer hypocotyls and inflorescences.
To compare the phenotypic effects resulting from the endogenous and
exogenous addition of BRs, the length of roots and hypocotyls of 16 seedlings
of
dwf4, wild-type controls, wild-type plants supplemented with 10-6 M epi-BL,
and two
independent AOD4 lines, grown for 12 days in the light or dark was measured.
As
described herein, dwf4-I displayed greatly reduced hypocotyl length both in
the light
and dark as compared to wild type. Wild-type roots are shortened when grown in
the
dark, but dwf4-1 root length was not significantly reduced in the dark
compared with
the reduction in hypocotyl length. When epi-BL is added, light-grown wild type
seedlings developed elongated hypocotyls, whereas roots were shorter than
untreated
control plants. These characteristic responses of wild-type plants to epi-BL
treatment
were similar in two independent AOD4 lines. The hypocotyl length of light-
grown
AOD4 seedlings was comparable to that of seedlings treated exogenously with
epi-
BL. However, dark-grown hypocotyls showed a dramatic increase in length as
compared to controls with or without epi-BL. Inhibition of root growth was
also
obvious in the AOD4 lines. Furthermore, the increased hypocotyl length and
reduced
root length were consistently observed in 15 independent transformants of
tobacco
(TOD4) harboring a pOD4 construct. This result suggests that the Arabidopsis
DWF4
enzyme also catalyzes BR biosynthesis in tobacco.
E. DWF4 overexpression results in increased plant height, bigger leaves, and
increased seed production
As shown below in Table 3, the effects of DWF4 overexpression on plant
growth were monitored during the course of development. The number of rosette
leaves at bolting was not significantly different between wild-type and AOD4
plants
(Table 3). The inflorescence height of wild type and two independent AOD4
lines
were comparable 20 days after germination (DAG). Later, the AOD4 lines outgrow
wild type. Surprisingly, AOD4 lines continue to grow beyond 35 DAG at the time
77
CA 02362603 2001-08-10
WO 00/47715 PCT/US00/03820
wild-type plants ceased elongation. At maturity, the height ofAOD4 lines was
135%
(AOD4-65) and 142% (AOD4-73) that of wild type, respectively. Similarly, TOD4
also displayed a 14% increase in plant height as compared to the control.
Interestingly, the increased inflorescence length in AOD4 plants seemed to be
at the
cost of stem stiffness. During development, AOD4 plants tend to fall over
earlier than
the Ws-2 wild type. In addition to plant height, comparison of rosette leaf
size
between wild type and AOD4 indicates that leaves, both rosette and cauline,
are
larger, especially in adult plants. TOD4 plants also possessed leaves that
were larger,
and had longer petioles relative to the control. Furthermore, additional
secondary
branches were found both in Arabidopsis and tobacco overexpression lines. In
AOD4
plants, this additional branching was associated with >2 times increased
number of
siliques per plant, leading to a 33 and 59% increase in seed production (Table
3). The
increased seed production in the AOD4 lines was mainly due to the increased
number
of seeds per plant than increase in the seed size, because the size was not
significantly
increased (Table 3). In addition to the increased number of seeds, the length
of silique
as well as the length of an internode between the first silique in a main
inflorescence
and the base of plant was increased (Table 3).
Figure 8 shows that stem growth is increased more than 20% compared to
wild type in DWF4 overexpression lines and Figure 9 shows that seed production
is
increased significantly over wild type in the DWF4-overexpressed lines. Figure
12
depicts hypocotyl length and root length in light and dark. Further, the
height of
AOD4 lines was greater than wild type over the days examined. In addition,
although
wild type plants ceased growth around five weeks after germination, AOD4
plants
continued to grow up to seven weeks.
78
CA 02362603 2001-08-10
WO 00/47715 PCT/US00/03820
N
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79
CA 02362603 2001-08-10
WO 00/47715 PCT/US00/03820
Thus, the DWF4 locus is defined by at least four mutant alleles. One of these
is the result of a T-DNA insertion. Plant DNA flanking the insertion site was
cloned
and used as a probe to isolate the entire DWF4 gene. Sequence analysis
revealed that
DWF4 encodes a cytochrome P450 monooxygenase with 43% identity to the putative
Arabidopsis steroid hydroxylating enzyme CONSTITUTIVE
PHOTOMORPHOGENESIS AND DWARFISM. Sequence analysis of two other
mutant alleles revealed deletions or a premature stop codon, confirming that
DWF4
had been cloned. This sequence similarity suggests that DWF4 functions in
specific
hydroxylation steps during BR biosynthesis. The dwarf phenotype can be rescued
with exogenously supplied brassinolide. dwf4 mutants display features of
light-regulatory mutants, but the dwarfed phenotype is entirely and
specifically
brassinosteroid dependent; no other hormone can rescue dwf4 to a wild-type
phenotype. Feeding studies utilizing BR intermediates showed that only
22a-hydroxylated BRs rescued the dwf4 phenotype, confirming that DWF4 acts as
a
22a-hydroxylase. In adult plants, strong GUS staining (indicative of dwf4
expression)
was found in the primordia of axilary inflorescences and secondary branches,
and in
young developing flowers. GUS expressing tissues correspond to the tissues
sensitive
to exogenously applied BRs leading to the hypothesis that these tissues are
putative
brassinolide biosynthetic sites. The inflorescence height of DWF4
overexpressing
lines increased >35% in Arabidopsis (AOD4) and 14% in tobacco (TOD4) as
compared to control plants at maturity. The total number of branches and
siliques
increased >2-fold in AOD4 plants, leading up to a 59% increase in seed
production.
The phenotypes of dwf4, DWF4, and AOD4 plants suggest that the degree of DWF4
transcription is associated with the degree of BR effects. In sum, it appears
be
possible to engineer agricultural plants with increased biomass and seed
yield.
CA 02362603 2001-08-10
WO 00/47715 PCT/US00/03820
SEQUENCE LISTING
<110> THE ARIZONA BOARD OF REGENTS ON BEHALF OF THE UNIVERSITY OF ARIZONA
<120> DWF4 POLYNUCLEOTIDES, POLYPEPTIDES AND USES THEREOF
<130> 2225-0001.40
<140>
<141>
<150> 60/119,657
<151> 1999-02-11
<150> 60/119,658
<151> 1999-02-11
<160> 18
<170> PatentIn Ver. 2.0
<210> 1
<211> 6888
<212> DNA
<213> Arabidopsis sp.
<400> 1
atgtgggtat tatattgttg ggttcggttt gagctacaat ataaatttcg tgtttctggt 60
tattctgttc acatgatttg agtttggttc tcaatttgga ttccaagata attaaatatt 120
aaaattcatt taaaatattt acaagtaatt aattatcttt acattgtatt gttataacaa 180
aatatctatc tttggtatat gagaaaatat ggagtttgga atttataata ataaaggaaa 240
taatcgattc catttggttg gattacacag ttaagttttt gtgtttcttt tgttatatgt 300
atatgagtaa atcaaaaaga gtattgattg aagtgtaaac atatttcgtt atgaccccca 360
aaaaaaaaaa aaaaacaaac aaacaaaccc cccccccgat atagtttttg gttctggatt 420
aggtttattt gatcataatt acatgcatca tttctttgat tactatgaag attttcttac 480
caattaaaat ttcgaattca tatctcttga ttattaaatt aaatacgagt gtgaatatcc 540
gtttatcgat cactccaatc atgattatga ttcttgtgct aatccagcaa attattaaca 600
agagtattga gaaaaaaccg aaaataagaa aagggaaaga gtagtgaccc atggagtatg 660
tgaataatta tcaaagagaa taagagatga caaccaaaag gttgtggaat aatggtccct 720
gccagctttc tctcacaatc aatatcgacc ctatttggat tttctggata ttcgttaaaa 780
tttgcgataa cgattgtgaa aaatatttta tttgttagct gatctcaata ttatgttcca 840
ggtatttgca taatcttctg tttaaagcat attttgtctt tctttttgtt tcgtttctct 900
taactatata ttatcgcgga tatatgataa caatgatata tcacaaaaca attgtctggg 960
accattttga ataaactttt tctcaaacat tacgggacac tggactcgac ccttaaaata 1020
cgattttaca gcgtcactag ttgagattac tagcataaag cataaaggac ccgttcaagc 1080
tatttataca aagttacaaa ctgaatatag cttgaaatcc tttagaaaat tttggaatta 1140
ccggttgtta tgtaaatata gatttagtgg taaacaaata tgttaatcaa ttagtggtca 1200
acatatacat aattccttac agaaaaaaca aacttaagag aagttaacat atccatatat 1260
gggtatgcta tacctttcac gtatgctata ctagagacta aagaatagtt atgtgatgtc 1320
gataaatgaa attcacacgc gtggtaataa ttatgggacc gtatgttacg atcactgcaa 1380
atatcattct tggttggtca acaataaaaa caaaaacaag aaaaaaagaa aacgattttt 1440
cttggattcc attcaatgat ctaaaatgca tagatctttt gggttacagt ttcgaagtcc 1500
tctacaagcg tgtaaccatc tgcaactatt aaattgcttt ctttaatgca tctttaacat 1560
atttattgtt agttggaatt taataagagc gaacttgtaa cattacaata tttatattag 1620
atactagtat gtgattattc caaatacata ctttggatgt ttaaacttaa tcttgtttct 1680
tcctacggta taaatattaa tcatcgaggt aaaaaaagtt ttgtcttatt ttcgcgatgc 1740
atgaaggata aacctaatga ctttaatttt ttgaaaatgt aaccctttta ctcatagatt 1800
aattaccgta tgtttttgtt gccataatga cagcctctac aactgtgata gtcaattttt 1860
1
CA 02362603 2001-08-10
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tctgcaaata ttaaattagg aattcaatgc tactatcaat agaagaaaca gctgagtatt 1920
acattttaat ttaaagacaa aatttttgaa aaatgttata atttctaaca atattattaa 1980
aatatgatgc ctataatgta tttcctatgt tcttaaaata ttttttttta tatttagtta 2040
taaatacatt atgaaccaat aatagttggt gaattcaaat atctccatta atattttttg 2100
aaatctacaa attattaata tttagtcaat aacaatgcat agaaagttcc aaaaaaaatt 2160
ttgttaacag aaacttccaa attttttttt tttatggaac aagaaataac agatagaaaa 2220
ctattttgtt gtggaatgga agtagtaata tacattaagc aaattttaaa aaattatata 2280
agcctatacg cgctcaaagt atgttatcta gtaggtgtaa ttaataatgc atggtgcgat 2340
tcagaattgg gacaacaatg aaaacggaat taaaatatta actttaaaat aaataaaaat 2400
ttgagtaaat gtgttttctg actattgagg ggcaaaaaaa agacaatgcc aaaagtctac 2460
gggtttgact gtccagttcg gtaataatct aataactctg tctttgaccg cacgctcgtg 2520
taggggtcct tctgacattt tcactgttct acccctactc gtgagcccac ccttttccca 2580
tatcctaagg gtaattttgg aaatcccaat ttaaaccgat tgagaccgta ccggacttcc 2640
tgggattctg ctggagcatt tatcaaaaat tattagcacg aatgggttta ttaatttaaa 2700
aactcacaac ttgatcagat aaaatttcat aaacactttt acgatggatt cgtacgatct 2760
atctaatgac tttttttttt ctaccacggt ggatgaaagt tatagtacta ttagccagag 2820
acaattgatt atagatatat ccattaatcc atgatattta tgatataaat agctgttaaa 2880
ctatttcagc atcgcagctt tctgcaactt ttgtttttaa tttaagagtt taataaataa 2940
aagtattaaa aggagcataa cgaggcaaca aaagtaatga acacggagaa acaaaagcca 3000
tgaagctcat tggttagttt aagcttaata agaagatttt attaaatttt aatgacgatg 3060
ataacaatta tattttctga cttctttaaa accccctctt acaaacagaa gctccctttt 3120
tcagtagaag tccgattccc aatcttaaag acaaagccat tagaaagaga aagtgagtga 3180
gagagagaga gaaactagct ccatgttcga aacagagcat catactctct tacctcttct 3240
tcttctccca tcgcttttgt ctcttcttct cttcttgatt ctcttgaaga gaagaaatag 3300
aaaaaccaga ttcaatctac ctccgggtaa atccggttgg ccatttcttg gtgaaaccat 3360
cggttatctt aaaccgtaca ccgccacaac actcggtgac ttcatgcaac aacatgtctc 3420
caagtaaaca acaacatctt ccaaaaactc aaaaaaataa atcctctgtt tttgaaattt 3480
gactaatgtt gtttatttta caggtatggt aagatatata gatcgaactt gtttggagaa 3540
ccaacgatcg tatcagctga tgctggactt aatagattca tattacaaaa cgaaggaagg 3600
ctctttgaat gtagttatcc tagaagtata ggtgggattc ttgggaaatg gtcgatgctt 3660
gttcttgttg gtgacatgca tagagatatg agaagtatct cgcttaactt cttaagtcac 3720
gcacgtctta gaactattct acttaaagat gttgagagac atactttgtt tgttcttgat 3780
tcttggcaac aaaactctat tttctctgct caagacgagg ccaaaaaggt ttttattttt 3840
atcttttatt ttgctaaatt tttttgttta tgaatcttta gagtttctaa cttttttttt 3900
tttaattgaa cagtttacgt ttaatctaat ggcgaagcat ataatgagta tggatcctgg 3960
agaagaagaa acagagcaat taaagaaaga gtatgtaact ttcatgaaag gagttgtctc 4020
tgctcctcta aatctaccag gaactgctta tcataaagct cttcaggtac atttattttt 4080
ttttgctgta aagtcacaaa ctctcattat aggtttttaa ttttatttta tgtgttaaat 4140
aaaatatcta aaatggttgt gtagtcacga gcaacgatat tgaagttcat tgagaggaaa 4200
atggaagaga gaaaattgga tatcaaggaa gaagatcaag aagaagaaga agtgaaaaca 4260
gaggatgaag cagagatgag taagagtgat catgttagga aacaaagaac agacgatgat 4320
cttttgggat gggttttgaa acattcgaat ttatcgacgg agcaaattct cgatctcatt 4380
cttagtttgt tatttgccgg acatgagact tcttctgtag ccattgctct cgctatcttc 4440
ttcttgcaag cttgccctaa agccgttgaa gagcttaggg taagataatt ataacagcac 4500
aagttaatta ctaccaaatt gttacgtatt atataagtta ttatagaatt attctattag 4560
aatatacgat gaaaaaagta tgtatattta attgtcacta attttatgtt tattgattta 4620
tacttttgaa ggaagagcat cttgagatcg cgagggccaa gaaggaacta ggagagtcag 4680
aattaaattg ggatgattac aagaaaatgg actttactca atgtgtatgt tactatcatt 4740
ctcattattt attctatgtt catatgattt atgatgaaac caaaattatt gatttttttt 4800
ttggtgtgtg tgaaggttat~aaatgaaact cttcgattgg gaaatgtagt taggtttttg 4860
catcgcaaag cactcaaaga tgttcggtac aaaggtaaaa ctttacgtac aaaattttta 4920
aataatgaaa tccggaatat tgaaatctta ttggatgaaa aatattaaaa taatttacat 4980
ttcttaatgt tggaaaaaag gatacgatat ccctagtggg tggaaagtgt taccggtgat 5040
ctcagccgta catttggata attctcgtta tgaccaacct aatctcttta atccttggag 5100
atggcaacag gtaaataaaa agtttctctc gttaactatc gaaaattagt gtatagtttt 5160
ttcatctatt gcatgaatag atacgtccta cgtgatttac ctatctatag atactatacg 5220
agaactatta atctggcaaa aactttttat tattattatc tttcaagtta gatcttaaca 5280
cgtcatggat cattgatcac atgaaagcat ataaattaaa aataagagag agaaagagac 5340
gtgttggtgt aagtgtacgt gaagacaatt aattagtagg atggtatgtc tttaatgacg 5400
2
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taggagctgc ctaaatattc ttataatcgt gaccgttgat ttattattag tcacggcttt 5460
gatacaattt aagatttgac ggacgatggt accacggctt tgacggatct cacacgcccg 5520
atgacttgta cgtgcgttag attctgccac gttgactggt tttaatactt agatttataa 5580
ctctattaat tataacaact atcaaatcgg cgaattagag aaatatacta tatagtatta 5640
ttatgattat tatgagataa tactttatga aataagataa taatggtagt catgatgtta 5700
tagtgagtgg ggaaggtaag aggtggtgag agatgattaa tgaccccacg tggtgtggtg 5760
ccaacaagca cgtgttcttc ttcctttttt cttcccaact tctttttttg ggggtttatt 5820
gtgatttata aaatcggttt gtcgtttttt tttgtgacga gcagcaaaac aacggagcgt 5880
catcgtcagg aagtggtagt ttttcgacgt ggggaaacaa ctacatgccg tttggaggag 5940
ggccaaggct atgtgctggt tcagagctag ccaagttaga aatggcagtg tttattcatc 6000
atctagttct taaattcaat tgggaattag cagaagatga tcaaccattt gcttttcctt 6060
ttgttgattt tcctaacggt ttgcctatta gggtttctcg tattctgtaa aaaaaaaaaa 6120
agatgaaagt atttttattc tcttcttttt tttttgataa ttttaaatca ttttttttgc 6180
ccaatgatat ataaaaattt ggataaataa tattattgga tattcgtttt ttagttcggg 6240
tttgagaaaa gggtttcgac tttcgaaagt ggacgatgta tatagattgg gagctaggtt 6300
gagtctttgg acatttgtat tggatgttgt tgattattag tgtcgacact attaaacctt 6360
aaatgggctt tctataaggc ccaattatat tacgattata acaaagtgac aacttttact 6420
tcgtttttga tccgaagcaa taacaaattg tcaaatacca aacacaagaa ttatgtaaac 6480
actcgtgtgt gtctagtggg aaatcattgg gctggagact gaacatcaga acacaagaaa 6540
cctgtcaatt atggatacac ctcctatgac ggtttccaaa ctttatcttg attcttatcg 6600
tgttacattg acacaaagag ttaggtgtca aaaggactaa atgaataaca atagctctca 6660
ggataagaag gttcataaaa tggtttcttt attttgagaa gaaagagaga ggagctttta 6720
ctgtttcttg ggtcctattc ctttaaatga gagggtttcg tttttacttc ttctatctca 6780
tcatctttag gatcctcttc tagacgagta aagtaatcct cgttaccaag caatggtctc 6840
atcttttgaa gacaggtctt ttccaagtcc tagttcaggc caaagctt 6888
<210> 2
<211> 513
<212> PRT
<213> Arabidopsis sp.
<400> 2
Met Phe Glu Thr Glu His His Thr Leu Leu Pro Leu Leu Leu Leu Pro
1 5 10 15
Ser Leu Leu Ser Leu Leu Leu Phe Leu Ile Leu Leu Lys Arg Arg Asn
20 25 30
Arg Lys Thr Arg Phe Asn Leu Pro Pro Gly Lys Ser Gly Trp Pro Phe
35 40 45
Leu Gly Glu Thr Ile Gly Tyr Leu Lys Pro Tyr Thr Ala Thr Thr Leu
50 55 60
Gly Asp Phe Met Gln Gln His Val Ser Lys Tyr Gly Lys Ile Tyr Arg
65 70 75 80
Ser Asn Leu Phe Gly Glu Pro Thr Ile Val Ser Ala Asp Ala Gly Leu
85 90 95
Asn Arg Phe Ile Leu Gln Asn Glu Gly Arg Leu Phe Glu Cys Ser Tyr
100 105 110
Pro Arg Ser Ile Gly Gly Ile Leu Gly Lys Trp Ser Met Leu Val Leu
115 120 125
Val Gly Asp Met His Arg Asp Met Arg Ser Ile Ser Leu Asn Phe Leu
130 135 140
3
CA 02362603 2001-08-10
WO 00/47715 PCT/US00/03820
Ser His Ala Arg Leu Arg Thr Ile Leu Leu Lys Asp Val Glu Arg His
145 150 155 160
Thr Leu Phe Val Leu Asp Ser Trp Gln Gln Asn Ser Ile Phe Ser Ala
165 170 175
Gln Asp Glu Ala Lys Lys Phe Thr Phe Asn Leu Met Ala Lys His Ile
180 185 190
Met Ser Met Asp Pro Gly Glu Glu Glu Thr Glu Gln Leu Lys Lys Glu
195 200 205
Tyr Val Thr Phe Met Lys Gly Val Val Ser Ala Pro Leu Asn Leu Pro
210 215 220
Gly Thr Ala Tyr His Lys Ala Leu Gln Ser Arg Ala Thr Ile Leu Lys
225 230 235 240
Phe Ile Glu Arg Lys Met Glu Glu Arg Lys Leu Asp Ile Lys Glu Glu
245 250 255
Asp Gln Glu Glu Glu Glu Val Lys Thr Glu Asp Glu Ala Glu Met Ser
260 265 270
Lys Ser Asp His Val Arg Lys Gln Arg Thr Asp Asp Asp Leu Leu Gly
275 280 285
Trp Val Leu Lys His Ser Asn Leu Ser Thr Glu Gln Ile Leu Asp Leu
290 295 300
Ile Leu Ser Leu Leu Phe Ala Gly His Glu Thr Ser Ser Val Ala Ile
305 310 315 320
Ala Leu Ala Ile Phe Phe Leu Gln Ala Cys Pro Lys Ala Val Glu Glu
325 330 335
Leu Arg Glu Glu His Leu Glu Ile Ala Arg Ala Lys Lys Glu Leu Gly
340 345 350
Glu Ser Glu Leu Asn Trp Asp Asp Tyr Lys Lys Met Asp Phe Thr Gln
355 360 365
Cys Val Ile Asn Glu Thr Leu Arg Leu Gly Asn Val Val Arg Phe Leu
370 375 380
His Arg Lys Ala Leu Lys Asp Val Arg Tyr Lys Gly Tyr Asp Ile Pro
385 390 395 400
Ser Gly Trp Lys Val Leu Pro Val Ile Ser Ala Val His Leu Asp Asn
405 410 415
Ser Arg Tyr Asp Gln Pro Asn Leu Phe Asn Pro Trp Arg Trp Gln Gln
420 425 430
Gln Asn Asn Gly Ala Ser Ser Ser Gly Ser Gly Ser Phe Ser Thr Trp
435 440 445
Gly Asn Asn Tyr Met Pro Phe Gly Gly Gly Pro Arg Leu Cys Ala Gly
450 455 460
4
CA 02362603 2001-08-10
WO 00/47715 PCT/US00/03820
Ser Glu Leu Ala Lys Leu Glu Met Ala Val Phe Ile His His Leu Val
465 470 475 480
Leu Lys Phe Asn Trp Glu Leu Ala Glu Asp Asp Gln Pro Phe Ala Phe
485 490 495
Pro Phe Val Asp Phe Pro Asn Gly Leu Pro Ile Arg Val Ser Arg Ile
500 505 510
Leu
<210> 3
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: primer D40VERF
<400> 3
atgttcgaaa cagagcatca tact 24
<210> 4
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: primer D4PRM
<400> 4
cctcgatcaa agagagagag a 21
<210> 5
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: primer D4RTF
<400> 5
ttcttggtga aaccatcggt tatcttaaa 29
<210> 6
<211> 26
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: primer D4RTR
<400> 6
tatgataagc agttcctggt agattt 26
<210> 7
<211> 21
CA 02362603 2001-08-10
WO 00/47715 PCT/US00I03820
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: primer D4F1
<400> 7
cgaggcaaca aaagtaatga a 21
<210> 8
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: primer D4R1
<400> 8
gttagaaact ctaaagattc a 21
<210> 9
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: primer D4F2
<400> 9
gattcttggc aacaaaactc tat 23
<210> 10
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: primer D4R2
<400> 10
ccgaacatct ttgagtgctt 20
<210> 11
<211> 26
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: primer D4F3
<400> 11
gtgtgaaggt tataaatgaa actctt 26
<210> 12
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
6
CA 02362603 2001-08-10
WO 00/47715 PCT/US00/03820
<223> Description of Artificial Sequence: primer D4R3
<400> 12
ggtttaatag tgtcgacact aata 24
<210> 13
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: primer D4F4
<400> 13
ccgatgactt gtacgtgcgt to 22
<210> 14
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: primer D4F5
<400> 14
gcgaagcata taatgagtat ggat 24
<210> 15
<211> 26
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: primer D4R5
<400> 15
gttggtcata acgagaatta tccaaa 26
<210> 16
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: primer D4XLINIT
<400> 16
taggatccag ctagtttctc tctctctct 29
<210> 17
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: primer T7
<400> 17
taatacgact cactataggg 20
7
CA 02362603 2001-08-10
WO 00/47715 PCT/US00103820
<210> 18
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: primer
D40VERFA
<400> 18
gaattctaga atgttcgaaa cagagcatca to 32
8
