Note: Descriptions are shown in the official language in which they were submitted.
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CAULIFLOWER FLORAL MERISTEM IDENTITY GENES
AND METHODS OF USING SAME
This work was supported by grant DCB-9018749
awarded by the National Science Foundation. The United
States Government has certain rights in this invention.
BACICGROUND OF THE INVENTION
FIELD OF THT+'. INVENTION
This invention relates generally to the field
of plant flowering and more specifically to genes
involved in the regulation of floweri.ng.
BACKGROUND INFORMATION
A flower is the reproductive structure of a -
flowering plant. Following fertilization, the ovary of
the flower becomes a fruit and bears seeds. As a
practical consequence, production of fruit and
seed-derived crops such as grapes, beans, corn, wheat and
rice is dependent upon flowering.
Early in the plant life cycle, vegetative
growth occurs, and roots, stems and leaves are formed.
During the later period of reproductive growth, flowers
as well as new shoots or branches develop. However, the
factors responsible for the transition from vegetative to
Vb reproductive growth, and the onset of flowering, are
poorly understood.
r
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A variety of external signals, such as length
of daylight and temperature, affect the time of
flowering. The time of flowering also is subject to
genetic controls that prevent young plants from flowering
.
prematurely. Thus, the pattern of genes expressed in a
plant is an important determinant of the time of
flowering.
Given these external signals and genetic
controls, a relatively fixed period of vegetative growth
precedes flowering in a particular plant species. The
length of time required for a crop to mature to flowering
limits the geographic location in which it can be grown
and can be an important determinant of yield. In
addition, since the time of flowering determines when a
plant is reproductively mature, the pace of a plant
breeding program also depends upon the length of time
required for a plant to flower.
Traditionally, plant breeding involves
generating hybrids of existing plants, which are examined
for improved yield_or quality. The improvement of
existing plant crops through plant breeding is central to
increasing the amount of food grown in the world since
the amount of land suitable for agriculture is limited.
For example, the development of new strains of wheat,
corn and rice through plant breeding has increased the
yield of these crops grown in underdeveloped countries
such as Mexico, India and Pakistan. Unfortunately, plant
breeding is inherently a slow process since plants must
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be reproductively mature before selective breeding can
proceed.
YI
For some plant species, the length of time
needed to mature to flowering is so long that selective
breeding, which requires several rounds of backcrossing
progeny plants with their parents, is impractical. For
example, perennial trees such as walnut, hickory, oak,
maple and cherry do not flower for several years after
planting. As a result, breeding of such plant species
for insect or disease-resistance or to produce improved
wood or fruit, for example, would require many years,
even if only a few rounds of selection were performed.
Methods of promoting early flowering can make
breeding of long generation plants such as trees
practical for the first time. Methods of promoting early
flowering also would be useful for shortening growth
periods, thereby broadening the geographic range in which
a crop such as rice, corn or coffee can be grown.
Unfortunately, methods for promoting early flowering in a
plant have not yet been described. Thus, there is a need
for methods that promote early flowering. The present
invention satisfies this need and provides related
advantages as well.
~
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STTMMARY OF THE INVENTION
The present invention provides a nucleic acid
molecule encoding a CAULIFLOWER (CAL) gene product. For
example, the invention "
provides a nucleic acid molecule
encoding Arabidopsis thaliana CAL and a nucleic acid
molecule encoding Brassica oleracea CAL.
The invention also provides a nucleic acid
molecule encoding a truncated CAL gene product. For
example, the invention provides a nucleic acid molecule
encoding the truncated Brassica oleracea var. botrytis
CAL gene product. The invention also provides a
nucleotide sequence that hybridizes under relatively
stringent conditions to a nucleic acid molecule encoding
a CAL gene product, a truncated CAL gene product, or a
complementary sequence thereto.
The invention further provides the Arabidopsis
thaliana CAL gene, Brassica oleracea CAL gene and
Brassica oleracea var. botrytis CAL gene. In addition,
the invention provides a nucleotide sequence that
hybridizes under relatively stringent conditions to the
Arabidopsis thaliana CAL gene, Brassica oleracea CAL gene
or Brassica oleracea var. botrytis CAL gene, or a
complementary sequence thereto.
~
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The invention also provides vectors, including
expression vectors, containing a nucleic acid molecule
encoding a CAL gene product. The invention further
provides a kit for converting shoot meristem to floral
= 5 meristem in an angiosperm and a kit for promoting early
flowering in an angiosperm.
In addition, the invention provides a CAL
polypeptide, such as the Arabidopsis thaliana CAL
polypeptide or the Brassica oleracea CAL polypeptide, as
well as an antibody that specifically binds a CAL
polypeptide. The invention further provides the
truncated Brassica oleracea var. botrytis CAL polypeptide
and an antibody that specifically binds the truncated
Brassica oleracea var. botrytis CAL polypeptide.
The invention further provides a method of
identifying a Brassica having a modified CAL allele by
detecting a polymorphism associated with a CAL locus,
where the CAL locus comprises a modified CAL allele that
does not encode an active CAL gene product. For example,
the polymorphism can be a restriction fragment length
polymorphism and the modified CAL allele can be the
Brassica oleracea var. botrytis CAL allele.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 illustrates the nucleotide (SEQ ID
NO: 1) and amino acid (SEQ ID NO: 2) sequence of the
Arabidopsis thaliana AP3 cDNA.
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Figure 2 illustrates the nucleotide (SEQ ID
NO: 3) and amino acid (SEQ ID NO: 4) sequence of the
Brassica oleracea API cDNA.
Figure 3 illustrates the nucleotide (SEQ ID =
NO: 5) and amino acid (SEQ ID NO: 6) sequence of the
Brassica oleracea var. botrytis AP1 cDNA.
Figure 4 illustrates the nucleotide (SEQ ID
NO: 7) and amino acid (SEQ ID NO: 8) sequence of the Zea
mays AP1 cDNA. The GenBank accession number is L46400.
Figure 5 illustrates the nucleotide (SEQ ID
NO: 9) and amino acid (SEQ ID NO: 10) sequence of the
Arabidopsis thaliana CAL cDNA.
Figure 6 illustrates the nucleotide (SEQ ID
NO: 11) and amino acid (SEQ ID NO: 12) sequence of the
Brassica oleracea CAL cDNA.
Figure 7 illustrates the nucleotide (SEQ ID
NO: 13) and amino acid (SEQ ID NO: 14) sequence of the
Brassica oleracea var. botrytis CAL cDNA.
Figure 8 illustrates CAL gene structure and
provides a comparison of various CAL amino acid
sequences.
Figure 8A. Exon-intron structure of
Arabidopsis CAL gene. Exons are shown as boxes and
introns as a solid line. Sizes (in base pairs) are
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7
indicated above. Locations of changes resulting in
mutant alleles are indicated by arrows. MADS and K
domains are hatched.
Figure 8B. An alignment of three deduced amino
acid sequences of CAL cDNAs. The complete Arabidopsis
thaliana CAL amino acid sequence is displayed. The
Brassica oleracea CAL (BoCAL) and Brassica oleracea var.
botrytis CAL (BobCAL) amino acid sequences are shown
directly below the Arabidopsis sequence where the
sequences differ. The API amino acid sequence is shown
for comparison. The MADS domain is amino acids 1-57 and
the K domain is underlined. GenBank accession numbers
are as follows: Arabidopsis thaliana CAL (L36925);
Brassica oleracea CAL (L36926) and Brassica oleracea var.
botrytis CAL (L36927).
Figure 9 illustrates the nucleotide (SEQ ID
NO: 15) and amino acid (SEQ ID NO: 16) sequence of the
Arabidopsis thaliana LEAFY (LFY) cDNA.
Figure 10 illustrates the genomic sequence of
Arabidopsis thaliana APi (SEQ ID NO: 17).
Figure 11 illustrates the genomic sequence of
Brassica oleracea AP1 (SEQ ID NO: 18).
Figure 12 illustrates the genomic sequence of
Brassica oleracea var. botrytis AP1 (SEQ ID NO: 19).
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Figure 13 illustrates the genomic sequence of
Arabidopsis thaliana CAL (SEQ ID NO: 20).
Figure 14 illustrates the genomic sequence of
Brassica oleracea CAL (SEQ ID NO: 21).
Figure 15 illustrates the genomic sequence of
Brassica oleracea var. botrytis CAL (SEQ ID NO: 22).
Figure 16 illustrates the nucleotide (SEQ ID
NO: 23) and amino acid (SEQ ID NO: 24) sequence of the
rat glucocorticoid receptor ligand binding domain.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a nucleic acid
molecule encoding a CAULIFLOWER (CAL) gene product, which
is a floral meristem identity gene product involved in
the conversion of shoot meristem to floral meristem. For
example, the invention provides a nucleic acid molecule
encoding Arabidopsis thaliana CAL and a nucleic acid
molecule encoding Brassica oleracea CAL (BoCAL) (Kempin
et al., Science, 267:522-525 (1995)).
As disclosed herein,
a CAL gene product can be expressed in an angiosperm,
thereby converting shoot meristem to floral meristem in
the angiosperm or promoting early flowering in the
ang:~osperm. The invention also provides a nucleic acid'
molecule encoding a truncated CAL gene product such as a
nucleic acid molecule encoding Brassica oleracea var.
boLrytis CAL (BobCAL). The invention also provides a
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nucleic acid molecule containing the Arabidopsis thaliana
CAL gene, a nucleic acid molecule containing the Brassica
oleracea CAL gene and a nucleic acid molecule containing
the Brassica oleracea var. botrytis CAL gene. The
invention further provides a kit for converting shoot
meristem to floral meristem and a kit for promoting early
flowering in an angiosperm. The invention provides a CAL
polypeptide and an antibody that specifically binds CAL
polypeptide. In addition, the invention provides the
truncated BobCAL polypeptide and an antibody that
specifically binds the truncated BobCAL polypeptide. The
invention further provides a method of identifying a
Brassica having a modified CAL allele by detecting a
polymorphism associated with a CAL locus, where the CAL
locus comprises a modified CAL allele that does not
encode an active CAL gene product.
The present invention provides a non-naturally
occurring angiosperm containing a first ectopically
expressible nucleic acid molecule encoding a first floral
meristem identity gene product. For example, the
invention provides a transgenic angiosperm containing a
first ectopically expressible floral meristem identity
gene product such as APETALAl (APl), CAULIFLOWER (CAL) or
LEAFY (LFY). Such a transgenic angiosperm can be, for
example, a cereal plant, leguminous plant, oilseed plant,
tree, fruit-bearing plant or ornamental flower.
A flower, like a leaf or shoot, is derived from
the shoot apical meristem, which is a collection of
' 30 undifferentiated cells set aside during embryogenesis.
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The production of vegetative structures, such as leaves
or shoots, and of reproductive structures, such as
flowers, is temporally segregated, such that a leaf or
shoot arises early in a plant life cycle, while a flower
5 develops later- The transition from vegetative to
reproductive development is the consecquence of a process
termed floral induction (Yanofsky, Ann. Rev. Plant
Phvsiol. Plant Mol, Biol. 46:167-188 (1995)).
Once induced, shoot apical meristem either
10 persists and produces floral meristem, which gives rise
to flowers, and lateral meristem, which gives rise to
branches, or is itself converted to floral meristem. The
fate of floral meristem is to differentiate into a single
flower having a fixed number of floral organs in a
whorled arrangement. Dicots, for example, contain four
whorls (concentric rings) in which sepals (first whorl)
and petals (second whorl) surround stamens (third whorl)
and carpels (fourth whorl).
Although shoot meristem and floral meristem
both consist of meristemic tissue, shoot meristem is
distinguishable from the more specialized floral
meristem. Shoot meristem generally is indeterminate and
gives rise to an unspecified number of floral and lateral
meristems. In contrast, floral meristem is determinate
and gives rise to the fixed number of floral organs that
comprise a flower.
By convention herein, a wild-type gene sequence
is represented in upper case italic letters (for example, `
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APE2'ALAI), and a wild-type gene product is represented in
upper case non-italic letters (APETALAl). Further, a
mutant gene allele is represented in lower case italic
letters (api), and a mutant gene product is represented
in lower case non-italic letters (apl).
Genetic studies have identified a number of
genes involved in regulating flower development. These
genes can be classified into different groups depending
on their function. Flowering time genes, for example,
are involved in floral induction and regulate the
transition from vegetative to reproductive growth. In
comparison, the floral meristem identity genes, which are
the subject matter of the present invention as disclosed
herein, encode proteins that promote the conversion of
shoot meristem to floral meristem. In addition, floral
organ identity genes encode proteins that determine
whether sepals, petals, stamens or carpels are formed
(Yanofsky, supra, 1995; Weigel, Ann. Rev. Genetics
29:19-39 (1995)). Some of the floral meristem identity
gene products also have a role in specifying organ
identity.
Floral meristem identity genes have been
identified by characterizing genetic mutations that
prevent or alter floral meristem formation. Among floral
meristem identity gene mutations in Arabidopsis thalzana,
those in the gene LEAFY (LFY) generally have the
strongest effect on floral meristem identity. Mutations
in LFY completely transform the basal-most flowers into
secondary shoots and have variable effects on
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later-arising (apical) flowers. In comparison, mutations
in the floral meristem identity gene APETALA1 _(AP1)
result in replacement of a few basal flowers by =
inflorescence shoots that are not subtended by leaves.
An apical flower produced in an api mutant has an
indeterminate structure in which a flower arises within a
flower. These mutant phenotypes indicate that both API
and LFY contribute to establishing the identity of the
floral meristem although neither gene is absolutely
required. The phenotype of 1fy api double mutants, in
which structures with flower-like characteristics are
very rare, indicates that LFY and API encode partially
redundant activities.
In addition to the LFY and AP1 genes, a third
locus that greatly enhances the api mutant phenotype has
been identified in Arabidopsis. This locus, designated
CAULXFLOWER (CAL), derives its name from the resulting
"cauliflower" phenotype, which is strikingly similar to
the common garden variety of cauliflower. In an apl cal
double mutant, floral meristem that develops behaves as
shoot meristem in that there is a massive proliferation
of meristems in the position that normally would be
occupied by a single flower. However, a plant homozygous
for a particular cal mutation (cal-1) has a normal
phenotype, indicating that AP1 can substitute for the
loss of CAL in these plants. In addition, because floral
meristem that forms in an api mutant behaves as shoot
meristem in an api cal double mutant, CAL can largely =
substitute for AP1 in specifying floral meristem. These
genetic data indicate that CAL and AP1 encode activities
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that are partially redundant in converting shoot meristem
to floral meristem.
Other genetic loci play at least minor roles in
specifying floral meristem identity. For example,
although a mutation in APETALA2 (AP2) alone does not
result in altered inflorescence characteristics, ap2 apl
double mutants have indeterminate flowers (fiowers with
shoot-like characteristics) (Bowman et al., Development
119:721-743 (1993)). Also, mutations in the CLAVATAI
(CLV1) gene result in an enlarged meristem and lead to a
variety of phenotypes (Clark et al., Development
119:397-418 (1993)). In a c1 vl api double mutant,
formation of flowers is initiated, but the center of each --
flower often develops as an indeterminate inflorescence.
Thus, mutations in CLAVATAI result in the loss of floral
meristem identity in the center of wild-type flowers.
Genetic evidence also indicates that the gene product of
UNUSUAL FLORAL ORGANS (UFO) plays a role in determining
the identity of floral meristem. Additional floral
meristem identity genes associated with altered floral
meristem formation remain to be isolated.
Mutations in another locus, designated TERMINAL
FLOWER (TFL), produce phenotypes that generally are
reversed as compared to mutations in the floral meristem
identity genes. For example, tf1 mutants flower early,
and the indeterminate apical and lateral meristems
develop as determinate floral meristems (Alvarez et al.,
Plant J. 2:103-116 (1992)). These characteristics
indicate that the TFL promotes maintenance of shoot
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meristem. TFL also acts directly or indirectly to
negatively regulate APl and LFY expression in shoot
meristem since APi and LFY are ectopically expressed in
the shoot meristem of tf1 mutants (Gustafson-Brown et
al., Cell 76:131-143 (1994); Weigel et al., Cell
69:843-859 (1992)). It is recognized that a plant having
a mutation in TFL can have a phenotype similar to a
non-naturally occurring angiosperm of the invention.
Such tfl mutants, however, are explicitly excluded from
the scope of the present invention.
The results of such genetic studies indicate
that several floral meristem identity gene products,
including AP1, CAL and LFY, act redundantly to convert
shoot meristem to floral meristem and that TFL acts
directly or indirectly to negatively regulate expression
of the floral meristem identity genes. As disclosed
herein, ectopic expression of a single floral meristem
identity gene product such as AP1, CAL or LFY is
sufficient to convert shoot meristem to floral meristem.
Thus, the present invention provides a non-naturally
occurring angiosperm that contains an ectopically
expressible nucleic acid molecule encoding a floral
meristem identity gene product, provided that such
ectopic expression is not due to a mutation in an
endogenous TERMINAL FLOWER gene.
As disclosed herein, an ectopically expressible
nucleic acid molecule encoding a floral meristem identity
gene product can be, for example, a transgene encoding a
floral meristem identity gene product under control of a
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heterologous gene regulatory element. In addition, such
an ectopically expressible nucleic acid molecule can be
an endogenous floral meristem identity gene coding
sequence that is placed under control of a heterologous
5 gene regulatory element. The ectopically expressible
nucleic acid molecule also can be, for example, an
endogenous floral meristem identity gene having a
modified gene regulatory element such that the endogenous
floral meristem identity gene is no longer subject to
10 negative regulation by TFL.
The term "ectopically expressible" is used
herein to refer to a gene transcript or gene product that
can be expressed in a tissue other than a tissue in which
it normally is produced. The actual ectopic expression
15 thereof -is dependent on various factors and can be
constitutive or inducible expression. As disclosed
herein, AP1, which normally is expressed in floral
meristem, is ectopically expressible in shoot meristem.
As disclosed herein, when a floral meristem identity gene
product such as AP1, CAL or LFY is ectopically expressed
in shoot meristem, the shoot meristem is converted to
floral meristem and early flowering can occur (see
Examples II, IV and V).
In particular, an ectopically expressible
nucleic acid molecule encpding a floral meristem identity
gene product can be expressed prior to the developmental
time at which the corresponding endogenous gene normally
is expressed. For example, an Arabidopsis plant grown
,
under continuous light conditions expresses API just
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prior to day 18, when normal flowering begins. However,
as disclosed herein, AP1 can be ectopically expressed in
shoot meristem earlier than day 18, resulting in early
conversion of shoot meristem to floral meristem and early
flowering. As shown in Example IID, a transgenic
Arabidopsis plant that ectopically expresses AP1 in shoot
meristem under control of a constitutive promoter flowers
earlier than the corresponding non-transgenic plant (day
as compared to day 18).
10 As used herein, the term "floral meristem
identity gene product" means a gene product that promotes
conversion of shoot meristem to floral meristem. As
disclosed herein, expression of a floral meristem
identity gene product such as APi, CAL or LFY in shoot
meristem can convert shoot meristem to floral meristem.
Furthermore, expression of a floral meristem identity
gene product in shoot meristem also can promote early
flowering (Examples IID, IVA and V). A floral meristem
identity gene product is distinguishable from a late
flowering gene product or an early flowering gene
product, which are not encompassed within the present
invention. In addition, reference is made herein to an
"inactive" floral meristem identity gene product, as
exemplified by BobCAL (see below). Expression of an
inactive floral meristem identity gene product in an
angiosperm does not result in the conversion of shoot
meristem to floral meristem in the angiosperm.
A floral meristem identity gene product can be,
for example, an AP1 gene product such as Arabidopsis AP1,
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which is a 256 amino acid gene product encoded by the API
cDNA sequence isolated from Arabidopsis thaliana
(Figure 5, SEQ ID NO: 2). The Arabidopsis AP1 cDNA
encodes a highly conserved MADS domain, which can
function as a DNA-binding domain, and a K domain, which
is structurally similar to the coiled-coil domain of
keratins and can be involved in_protein-protein
interactions.
In Arabidopsis, AP1 RNA is expressed in flowers
but is not detectable in roots, stems or leaves (Mandel
et al., Nature 360:273-277 (1992), which is incorporated
herein by reference). The earliest detectable expression
of API RNA is in young floral meristem at the time it
initially forms on the flanks of shoot meristem.
Expression of AP1 increases as the floral meristem
increases in size; no AP1 expression is detectable in
shoot meristem. In later stages of development, API
expression ceases in cells that will give rise to
reproductive organs (stamens and carpels), but is
maintained in cells that will give rise to
non-reproductive organs (sepals and petals; Mandel,
supra, 1992) .
As used herein, the term "APETALAI" or "AP1"
means a floral meristem identity gene product that is
characterized, in part, by having an amino acid sequence
that is related to the Arabidopsis APi amino acid
sequence shown in Figure 1(SEQ ID NO: 2) or to the Zea
mays AP1 amino acid sequence shown in Figure 4 (SEQ ID
NO: 8). In nature, APi is expressed in floral meristem.
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CAULIFLOWER (CAL) is another example of a
floral meristem identity gene product. As used herein,
the term "CAULIFLOWER" or "CAL" means a floral meristem
identity gene product that is characterized in part by
having an amino acid sequence that has at-least about 70
percent identity with the amino acid sequence shown in
Figure 5 (SEQ ID NO: 10) in the region from amino acid 1
to amino acid 160 or with the amino acid sequence shown
in Figure 6 (SEQ ID NO: 12) in the region from amino acid
1 to amino acid 160. In nature, CAL is expressed in
floral meristem.
The present invention provides a nucleic acid
molecule encoding a CAL, including, for example, the
Arabidopsis CAL cDNA sequence shown in Figure 5 (SEQ ID
NO: 9). As disclosed herein, CAL, like AP1, contains a
MADS domain and a K domain. The MADS domains of. CAL and
APi differ in only five of 56 amino acid residues, where
four of the five differences represent conservative amino
acid replacements. Over the entire sequence, the
Arabidopsis CAL and Arabidopsis APi sequences (SEQ ID
NOS: 10 and 2) are 76% identical and are 88% similar if
conservative amino acid substitutions are allowed.
Similar to the expression pattern of AP1, CAL
RNA is expressed in young floral meristem in Arabidopsis.
However, in contrast to API expression, which is high
throughout sepal and petal development, CAL expression is
low in these organs.
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LEAFY (LFY) is yet another example of a floral
meristem identity gene product. As used herein, the term
"LEAFY" or "LFY" means a floral meristem identity gene
product that is characterized in part by having an amino
acid sequence that is related to the amino acid sequence
shown in Figure 9 (SEQ ID NO: 16) In nature, LFY is
expressed in floral meristem as well as during vegetative
development. As disclosed herein, ectopic expression of
floral meristem identity gene products, which normally
are expressed in floral meristem, such as AP1 or CAL or
LFY or combinations thereof, in shoot meristem can
convert shoot meristem to floral meristem and promote
early flowering.
Flower development in Arabidopsis is recognized
in the art as a model for flower development in
angiosperms in general. Gene orthologs corresponding to
the Arabidopsis genes involved in the early steps of
flower formation have been identified in distantly
related plant species, and these gene orthologs show
remarkably similar RNA expression patterns. Mutations in
these genes also result in phenotypes that correspond to
the phenotype produced by a similar mutation in
Arabidopsis. For example, orthologs of the Arabidopsis
floral meristem identity genes API and LFY and the
Arabidopsis organ identity genes AGAMOUS, APETALA3 and
PISTILLATA have been isolated from monocots such as maize
and, where characterized, reveal the anticipated RNA
expression patterns and related mutant phenotypes.
(Schmidt et al., Plant Cell 5:729-737 (7-993); and Veit et
al., Plant Cell 5:1205-1215 (1993)).
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Furthermore, a gene
ortholog can be functionally interchangeable in that it
can function across distantly related species boundaries
(Mandel et al., Cell 71:133-143 (1992)).
5 Taken together, these
data suggest that the underlying mechanisms controlling
the initiation and proper development of flowers are
conserved across distantly related dicot and monocot
boundaries. Therefore, results obtained using
10 Arabidopsis can be predictive of results that can be
expected in other angiosperms.
Floral meristem identity genes in particular
are conserved throughout the plant kingdom. For example,
a gene ortholog of Arabidopsis API has been isolated from
15 Antirrhinum majus (snapdragon; Huijser et al., EMBO J.
11:1239-1249 (1992)).
As disclosed herein, an ortholog of
Arabidopsis API also has been isolated from Zea Mays
(maize; see Example IA). Similarly, gene orthologs of
20 Arabidopsis LFY have been isolated from Antirrhinum
majus, tobacco and poplar tree (Coen et al., Cell,
63:1311-1322 (1990); Kelly et al., Plant Cell 7:225-234
(1995); and Strauss et al., Molec. Breed 1:5-26 (1995)).
In addition, a mutatioii in the Antirrhinum AP1 ortholog
results in a phenotype ::imilar to the Arabidopsis api
mutant phenotype described above (Huijser et al., supra,
1992). Similarly, a mutation in the P.ntirrninum LFY
ortholog results in a phenotype similar to the
Arabidopsis lfy mutant phenotype (Coen et al., supra,
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1995). These studies indicate that AP1 and LFY function
similarly in distantly related angiosperms.
A floral meristem identity gene product also
can function across species boundaries. For example,
Arabidopsis LFY can convert shoot meristem to floral
meristem when expressed in aspen trees (Weigel and
Nilsson, Nature 377:495-500 (1995)).
As disclosed herein, a nucleic
acid molecule encoding an Arabidopsis AP1 or CAL gene
product (SEQ ID NOS: 1 and 9), for example, also can be
used to convert shoot meristem to floral meristem in an
angiosperm. Thus, a nucleic acid molecule encoding an
Arabidopsis AP1 gene product (SEQ ID NO: 1) or an
Arabidopsis CAL gene product (SEQ ID NO: 9) can be
introduced into an angiosperm such as corn, wheat or rice
and, upon expression, can convert shoot meristem to
floral meristem in the transgenic angiosperm.
Furthermore, as disclosed herein, the conserved nature of
an AP1 or CAL or LFY gene among diverse angiosperms,
allows a nucleic acid molecule encoding a floral meristem
identity gene product from essentially any angiosperm to
be introduced into essentially any other angiosperm,
wherein the expression of the nucleic acid molecule in
shoot meristem can convert shoot meristem to floral
meristem.
If desired, a novel API, CAL or LFY seauence
can be isolated from an angiosperm using a nucleotide
sequence as a probe and methods well known in the art of
molecular biology (Sambrook et al. (eds.), Molecular
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c'loning= A Laboratory Manual (Second Edition),
Plainview, NY: Cold Spring Harbor Laboratory Press
(198 ))=
As exemplified herein and discussed in detail below (see
S Example IA), the API ortholog from Zea Mays (maize; SEQ
ID NO: 7) was isolated using the Arabidopsis API cDNA as
a probe (SEQ ID NO: 1).
In one embodiment, the invention provides a
non-naturally occurring angiosperm that contains an
ectopically expressible nucleic acid molecule encoding a
floral meristem identity gene product and that is
characterized by early flowering. As used herein, the
term "characterized by early flowering," when used in
reference to a non-naturally occurring angiosperm of the
invention, means a non-naturally occurring angiosperm
that forms flowers sooner than flowers would form on a
corresponding naturally occurring angiosperm that does
not ectopically express a floral meristem identity gene
product, grown under the same conditions. Flowering
times for naturally occurring angiosperms are well known
in the art and depend, in part, on genetic factors and on
the environmental conditions, such as day length. Thus,
given a defined set of environmental conditions, a
naturally occurring plant will flower at a relatively
predictable time.
It is recognized that various transgenic p_ants
that are characterized by early flowering have been
described. Such transgenic plants are described herein
and are readily distinguishable or explicitly excluded
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from the present invention. For example, a product of a
"late-flowering gene" can promote early flowering but
does not specify the conversion of shoot meristem to
floral meristem. Therefore, a transgenic plant
expressing a late-flowering gene product is
distinguishable from a non-naturally occurring angiosperm
of the invention. For example, a transgenic plant
expressing the late-flowering gene, CONSTANS (CO),
flowers earlier than a corresponding wild type plant
(Putterill et al., Cell 80:847-857 (1995)). However,
expression of exogenous CONSTANS does not convert shoot
meristem to floral meristem.
Early flowering also has been observed in a
transgenic tobacco plant expressing an exogenous rice
MAIDS domain gene. Although the product of this gene
promotes early flowering, it does not specify the
identity of floral meristem and, thus, cannot convert
shoot meristem to floral meristem (Chung et al., Plant
Mol. Biol. 26:657-665 (1994)). Therefore, the
early-flowering CO and rice MADS domain gene transgenic
plants are distinguishable from the early-flowering
non-naturally occurring angiosperms of the invention.
Mutations in a class of genes known as
"early-flowering genes" also result in plants that flower
prematurely. Such early flowering genes include, for
example, EARLY FLOWERING 1-3 (ELF1, ELF2, ELF3);
EMBRYONIC FLOWER 1,2 (EMFZ, EMF2); LONG HYPOCOTYL 1,2
(HYI, HY2 ) ; PHYTOCHROME B (PHYB ) , SPINDLY (SPY) and
TERMINAL FLOWER (TFL) (Weigel, supra, 1995). However,
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the wild type product of an early flowering gene retards
flowering and is distinguishable from a floral meristem
identity gene product in that it does not promote
conversion of shoot meristem to floral meristem.
An Arabidopsis plant having a mutation in the
TERMINAL FLOWER (TFL) gene flowers early and is
characterized by the conversion of shoots to flowers
(Alvarez et al., Plant J. 2:103-116 (1992)).
However, TFL is not a
floral meristem identity gene product, as defined herein.
Specifically, it is the loss of TFL that promotes
conversion of shoot meristem to floral meristem. Since
the function of TFL is to antagonize formation of floral
meristem, a tfl mutant, which has lost this antagonist
function, permits conversion of shoot meristem to floral
meristem. Although TFL is not a floral meristem identity
gene product and does not itself convert shoot meristem
to floral meristem, the loss of TFL can result in a nlant
with an ectopically expressed floral meristem identity
gene product. Such tfl mutants, in which a mutation in
TFL results in conversion of shoot meristem to floral
meristem, are explicitly excluded from the present
invention.
As used herein, the term "non-naturally
occurring angiosperm" means an angiosperm that contains a
genome that has been modified by man. A transgenic
angiosperm, for example, contains an exogenous nucleic
acid molecule and, therefore, contains a genome tha-~ has
been modified by man. Furthermore, an angiosperm that
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contains, for example, a mutation in an endogenous floral
meristem identity gene regulatory element as a result of
exposure to a mutagenic agent by man also contains a
genome that has been modified by man. In contrast, a
plant containing a spontaneous or naturally occurring
mutation is not a "non-naturally occurring angiosperm"
and, therefore, is not encompassed within the invention.
As used herein, the term "transgenic" refers to
an angiosperm that contains in its genome an exogenous
nucleic acid molecule, which can be derived from the same
or a different species. The exogenous nucleic acid
molecule that is introduced into the angiosperm can be a
gene regulatory element such as a promoter or other
regulatory element or can be a coding sequence, which can
be linked to a heterologous gene regulatory element.
As used herein, the term "angiosperm" means a
flowering plant. Angiosperms are well known and produce
a variety of useful products including materials such as
lumber, rubber, and paper; fibers such as cotton and
linen; herbs and medicines such as quinine and
vinblastine; ornamental flowers such as roses and
orchids; and foodstuffs such as grains, oils, fruits and
vegetables.
Angiosperms are divided into two broad classes
based on the number of cotyledons, which are seed leaves
that generally store or absorb food. Thus, a
monocotyledonous angiosperm is an angiosperm having a
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single cotyledon, and a dicotyledonous angiosperm is an
angiosperm having two cotyledons.
.
Angiosperms encompass a variety of flowering
plants, including, for example, cereal plants, leguminous
plants, oilseed plants, trees, fruit-bearing plants and
ornamental flowers, which general classes are not
necessarily exclusive. Such angiosperms include for
example, a cereal plant, which produces an edible grain
cereal. Such cereal plants include, for example, corn,
rice, wheat, barley, oat, rye, orchardgrass, guinea
grass, sorghum and turfgrass. In addition, a leguminous
plant is an angiosperm that is a member of the pea family
(Fabaceae) and produces a characteristic fruit knownas a
legume. Examples of leguminous plants include, for
example, soybean, pea, chickpea, moth bean, broad bean,
kidney bean, lima bean, lentil, cowpea, dry bean, and
peanut. Examples of legumes further also include
alfalfa, birdsfoot trefoil, clover and sainfoin.
Furthermore, an oilseed plant is an angiosperm that has
seeds useful as a source of oil. Examples of oilseed
plants include soybean, sunflower, rapeseed and
cottonseed.
A tree is an angiosperm and is a perennial
woody plant, generally with a single stem (trunk).
Examples of trees include alder, ash, aspen, basswood
(linden), beech, birch, cherry, cottonwood, elm,
eucalyptus, hickory, locust, maple, oak, persimmon,
poplar, sycamore, walnut and willows. Such trees are
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used for pulp, paper, and structural material, as well as
providing a major source of fuel.
~
A fruit-bearing plant also is an angiosperm and
produces a mature, ripened ovary (usually containing
seeds) that is suitable for human or animal consumption.
Examples of fruit-bearing plants include grape, orange,
lemon, grapefruit, avocado, date, peach, cherry, olive,
plum, coconut, apple and pear trees and blackberry,
blueberry, raspberry, strawberry, pineapple, tomato,
cucumber and eggplant plants. An ornamental flower is an
angiosperm cultivated for its decorative flower.
Examples of ornamental flowers include rose, orchid,
lily, tulip and chrysanthemum, snapdragon, camelia,
carnation and petunia. The skilled artisan will
recognize that the invention can be practiced on these or
other angiosperms, as desired.
In various embodiments, the present invention
provides a non-naturally occurring angiosperm having an
ectopically expressible first nucleic acid molecule
encoding a first floral meristem identity gene product,
provided the first nucleic acid molecule is not
ectopically expressed due to a mutation in an endogenous
TFL gene. If desired, a non-naturally occurring
angiosperm of the invention can contain an ectopically
expressible second nucleic acid molecule encoding a
second floral meristem identity gene product, which is
different from the first floral meristem identity gene
product.
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An ectopically expressible nucleic acid
molecule can be expressed, as desired, either
constitutively or inducibly. Such an ectopically =
expressible nucleic acid molecule can be an endogenous
nucleic acid molecule and can contain, for example, a
mutation in its endogenous gene regulatory element or can
contain an exogenous, heterologous gene regulatory
element that is linked to and directs expression of the
endogenous nucleic acid molecule. In addition, an
ectopically expressible nucleic acid molecule encoding a
floral meristem identity gene product can be an exogenous
nucleic acid molecule encoding a floral meristem identity
gene product and containing a heterologous gene
regulatory element.
The invention provides, for example, a
non-naturally occurring angiosperm containing a first
ectopically expressible nucleic acid molecule encoding a
first floral meristem identity gene product. If desired,
a non-naturally occurring angiosperm of the invention can
contain a floral meristem identity gene having a modified
gene regulatory element and also can contain a second
ectopically expressible nucleic acid molecule encoding a
second floral meristem identity gene product, provided
that neither the first nor second ectopically expressible
nucleic acid molecule is ectopically expressed due to a
mutation in an endogenous TERMINAL FLOWER gene.
As used herein, the term "modified gene
regulatory element" means a regulatory element having a
mutation that results in ectopic expression in shoot
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meristem of the floral meristem identity gene regulated
by the gene regulatory element. Such a gene regulatory
element can be, for example, a promoter or enhancer
element and can be positioned 5' or 3' to the coding
sequence or within an intronic sequence of the floral
meristem identity gene. Such a modification can be, for
example, a nucleotide insertion, deletion or substitution
and can be produced by chemical mutagenesis using a
mutagen such as ethylmethane sulfonate (see Example IIIA}
or by insertional mutagenesis using a transposable
element. For example, a modified gene regulatory element
can be a functionally inactivated binding site for TFL or
a gene product regulated by TFL, such that modification
of the gene regulatory element results in ectopic
15. expression of the floral meristem identity gene product
in shoot meristem.
The invention also provides a transgenic
angiosperm containing a first exogenous gene promoter
that regulates a first ectopically expressible nucleic
acid molecule encoding a first floral meristem identity
gene product and a second exogenous gene promoter that
regulates a second ectopically expressible nucleic acid
molecule encoding a second floral meristem identity gene
product.
The invention also provides a transgenic
angiosperm containing a first exogenous ectopically
expressible nucleic acid molecule encoding a first floral
meristem identity gene product and a second exogenous
gene promoter that regulates a second ectopically
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expressible nucleic acid molecule encoding a second
floral meristem identity gene product, provided that the
first nucleic acid molecule is not ectopically expressed
due to a mutation in an endogenous TERMINAL FLOWER gene.
5 The invention also provides a transgenic
angiosperm containing a first exogenous ectopically
expressible nucleic acid molecule encoding a first floral
meristem identity gene product and a second exogenous
ectopically expressible nucleic acid molecule encoding a
10 second floral meristem identity gene product, where the
first floral meristem identity gene product is different
from the second floral meristem identity gene product and
provided that neither nucleic acid molecule is
ectopically expressed due to a mutation in an endogenous
15 TERMINAL FLOWER gene.
The ectopic expression of first and second
floral meristem identity gene products can be
particularly useful. For example, ectopic expression of
APl and LFY in a plant promotes flowering earlier than
20 ectopic expression of AP1 alone or ectopic expression of
LFY alone. Thus, plant breeding, for example, can be
further accelerated, if desired.
First and second floral meristem identity gene
products can be, for example, AP1 and CAL, or can be APl
25 and LFY or can be CAL and LFY. It should be recognized
that where a transgenic angiosperm of the invention
contains two exogenous nucleic acid molecules, the order
of introducing such a first and a second nucleic acid
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31
molecule is not important for purposes of the present
invention. Thus, a transgenic angiosperm of the
invention having, for example, APl as the first floral
meristem identity gene product and CAL as the second
floral meristem identity gene product is equivalent to a
transgenic angiosperm having CAL as the first floral
meristem identity gene product and APl as the second
floral meristem identity gene product.
The invention also provides methods of
converting shoot meristem to floral meristem in an
angiosperm by ectopically expressing an ectopically
expressible nucleic acid molecule encoding a floral
meristem identity gene product in the angiosperm. Thus,
the invention provides, for example, methods of
converting shoot meristem to floral meristem in an
angiosperm by introducing an exogenous ectopically
expressible nucleic acid molecule encoding a floral
meristem identity gene product into the angiosperm,
thereby producing a transgenic angiosperm. A floral
meristem identity gene product such as AP1, CAL or LFY,
or a chimeric protein containing, in part, a floral
meristem identity gene product (see below) is useful in
the methods of the invention.
As used herein, the term "introducing," when
used in reference to an angiosperm, means transferring an
exogenous nucleic acid molecule into the angiosperm. For
example, an exogenous nucleic acid molecule can be
introduced into an angiosperm by methods such as
Agrobacterium-mediated transformation or direct gene
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transfer methods including microprojectile-mediated
transformation (Klein et al., Nature 327:70-73 (1987)).
These and
other methods of introducing a nucleic acid molecule into
an angiosperm are well known in the art (Bowman et al.
(ed.), Arabidogsis: An Atlas of Morphology and
Development, New York: Springer (1994); Valvekens et
al., Proc. Natl. Acad. Sci., USA 85:5536-5540 (1988); and
Wang et al., Transformation of Plants and Soil
Microorganisms, Cambridge, UK: University Press (1995))=
As used herein, the term "converting shoot
meristem to floral meristem" means promoting the
formation of flower progenitor tissue where shoot
progenitor tissue would normally be formed. As a result
of the conversion of shoot meristem to floral meristem,
flowers form in an angiosperm where shoots normally would
form. The conversion of shoot meristem to floral
meristem can be identified using well known methods, such
as scanning electron microscopy, light microscopy or
visual inspection.
The invention also provides methods of
converting shoot meristem to floral meristem in an
angiosperm by introducing a first ectopically expressible
nucleic acid molecule encoding a first floral meristem
identity gene product and a second ectopically
expressible nucleic acid molecule encoding a second
floral meristem identity gene product into the
angiosperm. As discussed above, first and second floral
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meristem identity gene products useful in the invention
can be, for example, AP1 and CAL or AP1 and LFY or CAL
and LFY.
The invention also provides methods of
promoting early flowering in an angiosperm by ectopically
expressing a nucleic acid molecule encoding a floral
meristem identity gene product in the angiosperm,
provided that the nucleic acid molecule is not
ectopically expressed due to a mutation in an endogenous
TERMINAL FLOWER gene. For example, the invention
provides methods of promoting early flowering in an
angiosperm by introducing an ectopically expressible
nucleic acid molecule encoding a floral meristem identity
gene product into the angiosperm, thus producing a
transgenic angiosperm. A floral meristem identity gene
product such as APl, CAL or LFY, or a chimeric protein
containing, in part, a floral meristem identity gene
product (see below) is useful in methods of promoting
early flowering.
The present invention further provides nucleic
acid molecules encoding floral meristem identity gene
products. For example, the invention provides a nucleic
acid molecule encoding CAL, having at least about 70
percent amino acid identity with amino acids 1 to 160 of
SEQ ID NO: 10 or SEQ ID NO: 11. The invention also
provides a nucleic acid molecule encoding Arabidopsis
thaliana CAL having the amino acid sequence shown in
Figure 5 (SEQ ID NO: 10) and a nucleic acid molecule
encoding Brassica oleracea CAL having the amino acid
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sequence shown in Figure 6 (SEQ ID NO: 12). In addition,
the invention provides a nucleic acid molecule encoding
Brassica oZeracea AP1 having the amino acid sequence
shown in Figure 2 (SEQ ID NO: 4) and a nucleic acid
molecule encoding Brassica oleracea var. botrytis AP1
having the amino acid sequence shown in Figure 3 (SEQ ID
NO: 6). The invention also provides a nucleic acid
molecule encoding Zea mays AP1 having the amino acid
sequence shown in Figure 4 (SEQ ID NO: 8).
As disclosed herein, CAL is highly conserved
among different angiosperms. Forexample, Arabidopsis
CAL (SEQ ID NO: 10) and Brassica oleracea CAL (SEQ ID NO:
12) share about 80 percent amino acid identity. In the
region from amino acid 1 to amino acid 160, Arabidopsis
CAL and Brassica oleracea CAL are about 89 percent
identical at the amino acid level. Using a nucleotide
sequence derived from a conserved region of SEQ ID NO: 9
or SEQ ID NO: 11, a nucleic acid molecule encoding a
novel CAL ortholog can be isolated from other
angiosperms. Using methods sizch as those described by
Purugganan et al. (Genetics 40: 345-356 (1995)), one can
readily confirm that the newly isolated molecule is a CAL
ortholog. Thus, a nucleic acid molecule encoding CAL,
which has at least about 70 percent amino acid identity
with Arabidopsis CAL (SEQ ID NO: 10) or Brassica oleracea
CAL (SEQ ID NO: 12), can be isolated and identified using
well known methods.
The invention also provides a nucleic acid
molecule encoding a truncated CAL gene product. For
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example, the invention provides a nucleic acid molecule
encoding the Brassica oleracea var. botrytis CAL gene
product (BobCAL). BobCAL contains 150 amino acids of the
approximately 255 amino acids encoded by a full-length
5 CAL cDNA (see Figure 7; SEQ ID NO: 14; see, also, Figure
8B).
The invention also provides a nucleic acid
containing the Arabidopsis thaliana APi gene (Figure 10;
SEQ ID NO: 17), a nucleic acid molecule containing the
10 Brassica oleracea AP1gene (Figure 11; SEQ ID NO: 18) and
a nucleic acid molecule containing the Brassica oleracea
var. botrytis AP1 gene (Figure 12; SEQ ID NO: 19). In
addition, the invention also provides a nucleic acid
containing the Arabidopsis thaliana CAL gene (Figure 13;
15 SEQ ID NO: 20) and a nucleic acid molecule containing the
Brassica oleracea CAL gene (Figure 11; SEQ ID NO: 21).
In addition, the invention provides a nucleic acid
molecule containing the Brassica oleracea var. botrytis
CAL gene (Figure 15; SEQ ID NO: 22).
The invention further provides a nucleotide
sequence that hybridizes under relatively stringent
conditions to a nucleic acid molecule encoding a CAL, or
a complementary sequence thereof. In particular, such a
nucleotide sequence can hybridize under relatively
stringent conditions to a nucleic acid molecule encoding
Arabidopsis CAL (SEQ ID NO: 9) or Brassica oleracea CAL
(SEQ ID NO: 11), or a complementary sequence thereof.
Similarly, the present invention provides a nucleotide
sequencethat hybridizes under relatively stringent
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conditions to a nucleic acid molecule encoding Zea mays
AP1 (SEQ ID NO: 7), or a complementary sequence thereof.
In general, a nucleotide sequence that
hybridizes under relatively stringent conditions to a
nucleic acid molecule is a single-stranded nucleic acid
sequence that can range in size from about 10 nucleotides
to the full-length of a gene or a cDNA. Such a
nucleotide sequence can be chemically synthesized, using
routine methods or can be purchased from a commercial
source. Ih addition, such nucleotide sequences can be
obtained by enzymatic methods such as random priming
methods, the polymerase chain reaction (PCR) or by
standard restriction endonuclease digestion, followed by
denaturation (Sambrook et al., supra, 1989).
A nucleotide sequence that hybridizes under
relatively stringent conditions to a nucleic acid
molecule can be used, for example, as a primer for PCR
(Innis et al. (ed.) PCR Protocols: A Guide to Methods and
A.pplications, San Diego, CA: Academic Press, Inc.
(1990)). Such a nucleotide sequence generally contains
about 10 to about 50 nucleotides.
A nucleotide sequence that hybridizes under
relatively stringent conditions to a nucleic acid
molecule also can be used to screen a cDNA or genomic
library to obtain a related nucleotide sequence. For
example, a cDNA library that is prepared from rice or
wheat can be screened with a nucleotide sequence derived
from the Zea mays AP1 sequence in order to isolate a rice
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or wheat ortholog of AP1. Generally, such a nucleotide
sequence contains at least about 14-16 nucleotides
depending, for example, on the hybridization conditions
to be used.
A nucleotide sequence derived from a nucleic
acid molecule encoding Zea mays APi (SEQ ID NO: 7) also
can be used to screen a Zea mays cDNA library to isolate
a sequence that is related to but distinct from APi.
Furthermore, such a hybridizing nucleotide sequence can
be used to analyze RNA levels or patterns of expression,
as by northern blotting or by in situ hybridization to a
tissue section. Such a nucleotide sequence also can be
used in Southern blot analysis to evaluate gene structure
and identify the presence of related gene sequences.
One skilled in the art would select a
particular nucleotide sequence that hybridizes under
relatively stringent conditions to a nucleic acid
molecule encoding a floral meristem identity gene product
based on the application for which the sequence will be
used. For example, in order to isolate an ortholog of
AP1, one can choose a region of AP1 that is highly
conserved among known API sequences such as Arabidopsis
API (SEQ ID NO: 1) and Zea mays AP1 (GenBank accession
number L46400; SEQ ID NO: 7). Similarly, in order to
isolate an ortholog of CAL, one can choose a region of
CAL that is highly conserved among known CAL cDNAs, such
as Arabidopsis CAL (SEQ ID NO: 9) and Brassica CAL (SEQ
ID NO: 11). It further would be recognized, for example,
that the region encoding the MADS domain, which is common
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to a number of genes, can be excluded from the nucleotide
sequence. In addition, one can use a full-length
Arabidopsis AP1 or CAL cDNA nucleotide sequence (SEQ ID NO: 1 or SEQ ID NO: 9)
to isolate an ortholog of API or
CAL.
For example, the Arabidopsis API cDNA shown in
Figure 1 (SEQ ID NO: 1) can be used as a probe to
identify and isolate a novel APZ ortholog. Similarly,
the Arabidopsis CAL cDNA shown in Figure 5 (SEQ ID NO: 9)
can be used to identify and isolate a novel CAL ortholog
(see Examples IA and IIIC, respectively). In order to
identify related MADS domain genes, a nucleotide sequence
derived from the MADS domain of API or CAL, for example,
also can be useful to isolate a related gene sequence
encoding this DNA-binding motif.
Hybridization utilizing a nucleotide sequence
of the invention requires that hybridization be performed
under relatively stringent conditions such that
non-specific hybridization is minimized. Appropriate
hybridization conditions can be determined empirically,
or can be estimated based, for example, on the relative
G+C content of the probe and the number of mismatches
between the probe and target sequence, if known.
Hybridization conditions can be adjusted as desired by
varying, for example, the temperature of hybridizing or
the salt concentration (Sambrook, supra, 1989).
The invention also provides a vector containing
a nucleic acid molecule encoding a CAL gene product. In
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addition, the invention provides a vector containing a
nucleic acid molecule encoding the Zea mays AP1 gene
product. A vector can be a cloning vector or an
expression vector and provides a means to transfer an
exogenous nucleic acid molecule into a host cell, which
can be a prokaryotic or eukaryotic cell. Such vectors
are well known and include plasmids, phage vectors and
viral vectors. Various vectors and methods for
introducing such vectors into a cell are described, for
example, by Sambrook et al., supra, 1989, and by Glick
and Thompson (eds.), Methods in Plant Molecular Bioloqy
and Biotechnology, Boca Raton, FL: CRC Press (1993)).
The invention also provides an expression
vector containing a nucleic acid molecule encoding a
floral meristem identity gene product such as CAL, AP1 or
LFY. Expression vectors are well known in the art and
provide a means to transfer and express an exogenous
nucleic acid molecule into a host cell. Thus, an
expression vector contains, for example, transcription
start and stop sites such as a TATA sequence and a poly-A
signal sequence, as well as a translation start site such
as a ribosome binding site and a stop codon, if not
present in the coding sequence.
An expression vector can contain, for example,
a constitutive regulatory element useful for promoting
expression of an exogenous nucleic acid molecule in a
plant cell. The use of a constitutive regulatory element
can be particularly advantageous because expression from
CA 02215335 2006-09-21
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the element is relatively independent of developmentally
regulated or tissue-specific factors. For example, the
cauliflower mosaic virus 35S promoter (CaMV35S) is a
well-characterized constitutive regulatory element that
5 produces a high level of expression in all plant tissues
(Odell et al., Nature 313:810-812 (198-5)).
The CaMV35S promoter
is particularly useful because it is active in numerous
different angiosperms (Benfey and Chua, Science
10 250:959-966 (1990);
Odell et al., supra, 1985). Other
constitutive regulatory elements useful for expression in
an angiosperm include, for example, the nopaline synthase
(nos) gene promoter (An, Plant Physiol. 81:86 (1986)).
In addition, an expression vector of the
invention can contain a regulated gene regulatory element
such as a promoter or enhancer element. A particularly
useful regulated promoter is a tissue-specific promoter
such as the shoot meristem-specific CDC2 promoter
(Hemerly et al., Plant Cell 5:1711-1723 (1993)),
or the AGLB promoter,
which is active in the apical shoot meristem immediately
after the transition to f:owering (Mandel and Yanofsky,
Plant Cell 7:1763-1771 (1995)).
An expression vector of the invention also can
contain an inducible regulatory element, which has
conditional activity dependent upon the presence of a
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41
particular regulatory factor. Useful inducible
regulatory elements include, for example, a heat-shock
promoter (Ainley and Key, Plant Mol. Biol. 14:949 (1990)) or a
nitrate-inducible promoter derived from the spinach
nitrite reductase gene (Back et al., Plant Mol.
Bi . 17:9 (1991)).
A hormone-inducible element
(Yamaguchi-Shinozaki et al., Plant Mol. Biol. 15:905
(1990) and Kares et al., Plant Mol. Biol. 15:225 (1990)) or a
light-inducible promoter, such as that associated with
the small subunit of RuBP carboxylase or the LHCP gene
families (Feinbaum et al., MQl. Gen. Genet. 226:449
(1991) and Lam and Chua, Science 248:471
(1990)) also can be useful
in an expression vectcr of the invention. A human
glucocorticoid response element also can be used to
achieve steroid hormone-dependent gene expression in
plants (Schena et al., Proc. Natl. Acad. Sci. USA
88:10421 (1992.)).
An appropriate gene regulatory element such as
a promotor is selected depending on the desired pattern
or level of expression of a nucleic acid molecule linked
thereto. For example, a constitutive promoter, which is
active in all tissues, would be appropriate to express a
desired gene product in all cells containing the vector.
In addition, it can be desirable to restrict expression
of a nucleic acid molecule to a particular tissue or
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during a particular stage of development. A
developmentally regulated or tissue-specific expression
can be useful for this purpose and can avoid potential undesirable side-
effects that can accompany unregulated
expression. Inducible expression also can be
particularly useful to manipulate the timing of gene
expression such that, for example, a population of
transgenic angiosperms of the invention that contain an
expression vector comprising a floral meristem identity
gene linked to an inducible promoter can be induced to
flower essentially at the same time. Such timing of
flowering can be useful, for example, for manipulating
the time of crop harvest.
The invention also provides a kit containing an
expression vector having a nucleic acid molecule encoding
a floral meristem identity gene product. Such a kit is
useful for converting shoot meristem to floral meristem
in an angiosperm or for promoting early flowering in an
angiosperm. If desired, such a kit can contain
appropriate reagents, which can allow relatively high
efficiency of transformation of an angiosperm with the
vector. Furthermore, a control plasmid lacking the
floral meristem identity gene can be included in the kit
to determine, for example, the efficiency of
transformation.
The invention further provides a host cell
containing a vector comprising a nucleic acid molecule
encoding CAL. A host cell can be prokaryotic or
eukaryotic and can be, for example, a bacterial cell,
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yeast cell, insect cell, xenopus cell, mammalian cell or
plant cell.
The invention also provides a transgenic garden
} variety cauliflower plant containing an exogenous nucleic
acid molecule selected from the group consisting of a
nucleic acid molecule encoding a CAL gene product and a
nucleic acid molecule encoding an APi gene product. Such
a transgenic cauliflower plant can produce an edible
flower in place of the typical cauliflower vegetable.
A nucleic acid encoding CAL has been isolated
from a Brassica oleracea line that produces wild-type
flowers (BoCAL) and from the common garden variety of
cauliflower, Brassica oleracea var. botrytis (BobCAL)
which lacks flowers. The Brassica oleracea CAL cDNA (SEQ
ID NO: 10) is highly similar to the Arabidopsis CAL cDNA
(SEQ ID NO: 12; and see Figure 8). In contrast, the
Brassica oleracea var. botrytis CAL cDNA contains a stop
codon, predicting that the BobCAL protein will be
truncated after amino acid 150 (SEQ ID NO: 14 and see
Figure 8). The correlation of full-length Arabidopsis
and Brassica oleracea CAL gene products with a flowering
phenotype indicates that transformation of non-flowering
garden varieties of cauliflower such as Brassica oleracea
var. botrytis with a full-length CAL cDNA can induce
flowering in the transgenic cauliflower plant.
As used herein, the term "CAL gene product"
means a full-length CAL gene product that does not
terminate substantially before amino acid 255 and that,
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when ectopically expressed in shoot meristem, converts
shoot meristem to floral meristem. A nucleic acid
molecule encoding a CAULIFLOWER gene product can be, for
example, a nucleic acid molecule encoding Arabidopsis CAL
shown in Figure 5 (SEQ ID NO: 9) or a nucleic acid molecule encoding Brassica
oleracea CAL shown in Figure 6
(SEQ ID NO: 11). In comparison, a nucleic acid molecule
encoding a truncated CAL gene product that terminates
substantially before amino acid 255, such as the encoded
truncated BobCAL gene product (SEQ ID NO: 13), is not a
nucleic acid molecule encoding a CAL gene product as
defined herein. Furthermore, ectopic expression of
BobCAL in an angiosperm does not result in conversion of
shoot meristem to floral meristem.
As used herein, the term "APi gene product"
means a full-length API gene product that does not
terminate substantially before amino acid 256. A nucleic
acid molecule encoding an APi gene product can be, for
example, a nucleic acid molecule encoding Arabidopsis AP1
shown in Figure 1 (SEQ ID NO: 1), Brassica oleracea API
shown in Figure 2, (SEQ ID NO: 3), Brassica oleracea var.
botrytis API shown in Figure 3 (SEQ ID NO: 5) or Zea mays
API shown in Figure 4 (SEQ ID NO: 7).
The invention provides a CAL polypeptide having
at least about 70 percent amino acid identity with amino
acids 1 to 160 of SEQ ID NO: 10 or SEQ ID NO: 12. For
example, the Arabidopsis thaliana CAL polypeptide, having
the amino acid sequence shown as amino acids 1 to 255 in
Figure 5(SEQ ID NO: 10), and the Brassica oleracea CAL
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polypeptide, having the amino acid sequence shown as
amino acids 1 to 255 in Figure 6 (SEQ ID NO: 12) are
provided by the invention.
The invention also provides the truncated
5 Brassica oleracea var. botrytis CAL polypeptide having
the amino acid sequence shown as amino acids 1 to 150 in
Figure 7 (SEQ ID NO: 14). The BobCAL polypeptide can be
useful as an immunogen to produce an antibody that
specifically binds the truncated BoCAL polypeptide, but
10 does not bind a full length CAL gene product. Such an
antibody can be useful to distinguish between a full
length CAL and truncated CAL.
The invention provides also provides a Zea mays
AP1 polypeptide. As used herein, the term "polypeptide"
15 is used in its broadest sense to include proteins,
polypeptides and peptides, which are related in that each
consists of a sequence of amino acids joined by peptide
bonds. For convenience, the terms "polypeptide,"
"protein" and "gene product" are used interchangeably.
20 While no specific attempt is made to distinguish the size
limitations of a protein and a peptide, one skilled in
the art would understand that proteins generally consist
of at least about 50 to 100 amino acids and that peptides
generally consist of at least two amino acids up to a few
25 dozen amino acids. The term polypeptide is used
generally herein to include any such amino acid sequence.
The term polypeptide also includes an active
fragment of a floral meristem identity gene product. As
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used herein, the term "active fragment," means a
polypeptide portion of a floral meristem identity gene
product that can convert shoot meristem to floral
meristem or can provide early flowering. For example, an
active fragment of a CAL polypeptide can consist of an
amino acid sequence derived from a CAL protein as shown
in Figure 5 or 6 (SEQ ID NOS: 10 and 12) and that has an
activity of a CAL. An active fragment can be, for
example, an amino terminal or carboxyl terminal truncated
form of Arabidopsis thaliana CAL or Brassica oleracea CAL
(SEQ ID NOS: 10 or 12, respectively). Such anactive
fragment can be produced using well known recombinant DNA
methods (Sambrook et al., supra, 1989). The product of
the BobCAL gene, which is truncated at amino acid 150,
lacks activity in converting shoot meristem to floral
meristem and, therefore, is an example of a polypeptide
portion of a CAL floral meristem identity gene product
that is not an "active fragment."
An active fragment of a floral meristem
identity gene product can convert shoot meristem to
floral meristem and is readily identified using the
methods described in Example II, below). Briefly,
Arabidopsis can be transformed with a nucleic acid
molecule encoding a portion of a-floral meristem identity
gene product, in order to determine whether the fragment
can convert shoot meristem to floral meristem or promote
early flowering and, therefore, has an activity of a
floral meristem identity gene product.
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The invention further provides an antibody that
specifically binds a CAL polypeptide, an antibody that
specifically binds the truncated Brassica oleracea var.
botrytis CAL polypeptide, and an antibody that
specifically binds the Zea mays AP1 polypeptide. As used
herein, the term "antibody" is used in its broadest sense
to include polyclonal and monoclonal antibodies, as well
as polypeptide fragments of antibodies that retain a
specific binding activity for CAL protein of at least
about 1 x 105 M-1. One skilled in the art would know that
anti-CAL antibody fragments such as Fab, F(ab')z and Fv
fragments can retain specific binding activity for CAL
and, thus, are included within the definition of an
antibody. In addition, the term "antibody" as used
herein includes naturally occurring antibodies as well as
non-naturally occurring antibodies and fragments that
have binding activity such as chimeric antibodies or
humanized antibodies. Such non-naturally occurring
antibodies can be constructed using solid phase peptide
synthesis, produced recombinantly or obtained, for
example, by screening combinatorial libraries consisting
of variable heavy chains and variable light chains as
described by Huse et al., Science 246:1275-1281 (1989)).
An antibody "specific for" a polypeptide, or
that "specifically binds" a polypeptide, binds with
substantially higher affinity to that polypeptide than to
an unrelated polypeptide. An antibody specific for a
polypeptide also can have specificity for a related
polypeptide. For example, an antibody specific for
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Arabidopsis CAL also can have specificity for Brassica
oleracea CAL.
An anti-CAL antibody, for example, can be
prepared using a CAL fusion protein or a synthetic
peptide encoding a portion of Arabidopsis CAL or of
Brassica oleracea CAL as an immunogen. One skilled in
the art would know that purified CAL protein, which can
be prepared from natural sources or produced
recombinantly, or fragments of CAL, including a peptide
portion of CAL such as a synthetic peptide, can be used
as an immunogen. Non-immunogenic fragments or synthetic
peptides of CAL can be made immunogenic by coupling the
hapten to a carrier molecule such as bovine serum albumin
(BSA) or keyhole limpet hemocyanin (KLH). In addition,
various other carrier molecules and methods for coupling
a hapten to a carrier molecule are well known in the art
and described, for example, by Harlow and Lane,
Antibodies; A laboratory manual (Cold Spring Harbor
Laboratory Press, 1988).
An antibody that specifically binds the
truncated Bob CAL polypeptide or an antibody that
specifically binds the Zea mays APl polypeptide similarly
can be produced using such methods. An antibody that
specifically binds the truncated Brassica oleracea var.
botrvtis CAL polypeptide can be particularly useful to
distinguish between full-length CAL polypeptide and
truncated CAL polypeptide.
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The invention provides a method of ideritifying
a Brassica having a modified CAL allele by detecting a
polymorphism associated with a CAL locus, where the CAL
locus comprises a modified CAL allele that does not
encode an active CAL gene product. Such a method is
useful for the genetic improvement of Brassica plants, a
genus of_great economic value.
Brassica plants are a highly diverse group of
crop plants useful as vegetables and as sources of
condiment mustard, edible and industrial oil, animal
fodder and green manure. Brassica crops encompass a
variety of well known vegetables including cabbage,
cauliflower, broccoli, collard, kale, mustard greens,
Chinese cabbage and turnip, which can be interbred for
crop improvement (see, for example, King, Euphytica
50:97-112 (1990) and Crisp and Tapsell, Genetic
improvement of vecretable crops pp. 157-178 (199:)).
Breeding of Brassica crops is useful, for
example, for improving the quality and early development
of vegetables. In addition, such breeding can be useful
to increase disease resistance, such as resistance, of a
Brassica to clubroot disease or mildew; viral resistance,
such as resistance to turnip mosaic virus and cauliflower
mosaic virus; or pest resistance (King, supra, 1990).
The use of polymorphic molecular markers in the
breeding of Brassicae is well recognized in the art
(Crisp and Tapsell, supra, 1993). Identification of a
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polymorphic molecular marker that is associated with a
desirable trait can vastly accelerate the time required
to breed the desirable trait into a new Brassica species
or variant. In particular, since many rounds of
5 backcrossing are required to breed a new trait into a
different genetic background, early detection of a
desirable trait by molecular methods can be performed
prior to the time a plant is fully mature, thus
accelerating the rate of crop breeding (see, for example,
10 Figidore et al., Euphytica 69: 33-44 (1993)).
A polymorphism associated with a CAL locus
comprising a modified CAL allele that does not encode an
active CAL gene product, is disclosed herein. Figure 6
15 shows the nucleotide (SEQ ID NO: 11) and amino acid (SEQ
ID NO: 12) sequence of Brassica oleracea CAL (BoCAL), and
Figure 7 shows the nucleotide (SEQ ID NO: 13) and amino
acid (SEQ ID NO: 14) sequence of Brassica oleracea var.
botrytis CAL (BobCAL). At amino acid 150, which is
20 glutamic acid (Glu) in BoCAL, a stop codon is present in
BobCAL. This polymorphism results in a truncated BobCAL
gene product that is not active as a floral meristem
identity gene product. The BoCAL nucleic acid sequence
(ACGAGT) can be readily distinguished from the BobCAL
25 nucleic acid sequence (ACTAGT) using well known molecular
methods. For example, the polymorphic ACTAGT BobCAL
sequence is recognized by a SpeI restriction endonuclease
site, whereas the ACGAGT BoCAL sequence is not recognized
by SpeI. Thus, a restriction fragment length
30 polymorphism (RFLP) in BobCAL provides a simple means for
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identifying a modified CAL allele (BobCAL) and,
therefore, can serve as a marker to predict the
inheritance of the "cauliflower" phenotype.
A modified CAL allele encoding a truncated CAL
gene product also can serve as a marker to predict the
"cauliflower" phenotype in other cauliflower variants.
For example, nine romanesco variants of Brassica oleracea
var. botrytis, which each have the "cauliflower"
phenotype, were examined for the presence of a stop codon
at position 151 of the CAL coding sequence. All nine of
the romanesco variants contained the SpeI site that
indicates a stop codon and, thus, a truncated CAL gene
product. In contrast, Brassica oleracea variants that
lack the "cauliflower" phenotype (broccoli and brussels
sprouts) were examined for the SpeI site. In every case,
the broccoli and brussel sprout variants had a
full-length CAL coding sequence, as indicated by the
absence of the distinguishing Spel site. Thus, a
truncated CAL gene product can be involved in the
"cauliflower phenotype" in numerous different Brassica
variants.
As used herein, the term "modified CAL allele"
means a CAL allele that does not encode a CAL gene
product active in converting shoot meristem to floral
meristem. A modified CAL allele can have a modification
within a gene regulatory element such that a CAL gene
product is not produced. In addition, a modified CAL
allele can have a modification such as a mutation,
deletion or insertion in a CAL coding sequence which
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results in an inactive CAL gene product. For example, an
inactive CAL gene product can result from a mutation
creating a stop codon, such that a truncated, inactive
CAL gene product lacking the ability to convert shoot
meristem to floral meristem is produced.
As used herein, the term "associated" means
closely linked and describes the tendency of'two genetic
loci to be inherited together as a result of their
proximity. If two genetic loci are associated and are
polymorphic, one locus can serve as a marker for the
inheritance of the second locus. Thus, a polymorphism
associated with a CAL locus comprising a modified CAL
allele can serve as a marker for inheritance of the
modified CAL allele. An associated polymorphism can be
located in proximity to a CAL gene or can be located
within a CAL gene.
A polymorphism in a nucleic acid sequence can
be detected by a variety of methods. For example, if the
polymorphism occurs in a particular restriction
endonuclease site, the polymorphism can be detected by a
difference in restriction fragment length observed
following restriction with the particular restriction
endonuclease and hybridization with a nucleotide sequence
that is complementary to a nucleic acid sequence
including a polymorphism.
The use of restriction fragment length
polymorphism as an aid to breeding Brassicae is well
known in the art (see, for example, Slocum et al., Theor.
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53
Appl. Genet. 80:57-64 (1990); Kennard et al., Theor,
Appl. Genet. 87:721-732 (1994); and Figidore et al.,
supra, 1993)).
A restriction endonuclease such as SpeI,
which is useful for identifying the presence of a BobCAL
allele in an angiosperm, is readily available and can be
purchased from a commercial source. Furthermore, a
nucleotide sequence that is complementary to a nucleic
acid sequence having a polymorphism associated with a CAL
locus comprising a modified CAL allele can be derived,
for example, from the nucleic acid molecule encoding
Brassica oleracea var. botrytis CAL shown in Figure 7
(SEQ ID NO: 13) or from the nucleic acid molecule
encoding Brassica oleracea CAL shown in Figure 6 (SEQ ID
NO: 11).
In some cases, a polymorphism is not
distinguishable by a RFLP, but nevertheless can be used
to identify a Brassica having a modified CAL allele. For
example, the polymerase chain reaction (PCR) can be used
to detect a polymorphism associated with a CAL locus
comprising a modified CAL allele. Specifically, a
polymorphic region of a modified allele can be
selectively amplified by using a primer that matches the
nucleotide sequence of one allele of a polymorphic locus,
but does not match the sequence of the second allele
(Sobral and Honeycutt, The Polymerase Chain Reaction, pp.
304-319 (199,1)).
Other well-known approaches for analyzing a
polymorphism using PCR include discriminant hybridization
of PCR-amplified DNA to allele-specific oligonucleotides
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and denaturing gradient gel electrophoresis (see Innis et
al., supra, 1990).
The invention further provides a nucleic acid
molecule encoding a chimeric protein, comprising a
nucleic acid molecule encoding a floral meristem identity
gene product such as AP1, LFY or CAL operably linked to a
nucleic acid molecule encoding a ligand binding domain.
Expression of a chimeric protein of the invention in an
angiosperm is particularly useful because the ligand
binding domain confers regulatable activity on a gene
product such as a floral meristem identity gene product
to which it is fused. Specifically, the floral meristem
identity gene product component of the chimeric protein
is inactive in the absence of the particular ligand,
whereas, in the presence of ligand, the ligand binds the
ligand binding domain, resulting in floral meristem
identity gene product activity.
A nucleic acid molecule encoding a chimeric
protein of the invention contains a nucleic acid molecule
encoding a floral meristem identity gene product, such as
a nucleic acid molecule encoding the amino acid sequence
shown in Figure 1(SEQ ID NO: 2), in Figure 5(SEQ ID NO:
10), or in Figure 9 (SEQ ID NO: 10), either of which is
operably linked to a nucleic acid molecule encoding a
ligand binding domain. The expression of such a nucleic
acid molecule results in the production of a chimeric
protein comprising a floral meristem identity gene
product fused to a ligand binding domain. Thus, the
invention also provides a chimeric protein comprising a
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floral meristem identity gene product fused to a ligand
binding domain.
A ligand binding domain useful in a chimeric
protein of the invention can be a steroid binding domain
5 such as the ligand binding domain of a glucocorticoid
receptor, estrogen receptor, progesterone receptor,
androgen receptor, thyroid receptor, vitamin D receptor
or retinoic acid receptor. A particularly useful ligand
binding domain is a glucocorticoid receptor ligand
10 binding domain, encompassed, for example, within amino
acids 512 to 795 of the rat glucocorticoid receptor as
shown in Figure 16 (SEQ ID NO: 24; Miesfeld et al., Cell
46:389-399 (1986)).
15 A chimeric protein containing a ligand binding
domain, such as the rat glucocorticoid receptor ligand
binding domain, confers glucocorticoid-dependent activity
on the chimeric protein. For example, the activity of
chimeric proteins consisting of adenovirus ElA, c-myc,
20 c-fos, the HIV-1 Rev transactivator, MyoD or maize
regulatory factor R fused to the rat glucocorticoid
receptor ligand binding domain is regulated by
glucocorticoid hormone (Eilers et al., Nature 340:66
(1989); Superti-Furga et al., Proc. Natl. Acad. Sci.,
25 U.S.A. 88:5114 (1991); Hope et al., Proc. Natl. Acad.
Sc=., U.S.A. 87:7787 (1990); Hollenbera et al., Proc.
Nati Acad Sci U S A 90:8028 ,199;)).
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Such a chimeric protein also can be regulated
in plants. For example, a chimeric protein containing a
heterologous protein fused to a rat glucocorticoid
receptor ligand binding domain (amino acids 512 to 795)
was expressed under the control of the constitutive
cauliflower mosaic virus 35S promoter in Arabidopsis.
The activity of the chimeric protein was inducible; the
chimeric protein was inactive in the absence of ligand,
and became active upon treatment of transformed plants
with a synthetic glucocorticoid, dexamethasone (Lloyd et
al., Science 266:436-439 (1994)).
As disclosed herein, a ligand
binding domain fused to a floral meristem identity gene
product can confer ligand inducibility on the activity of
a fused floral meristem identity gene product in plants
such that, upon exposure to a particular ligand, the
floral meristem identity gene product is active.
Methods for constructing a nucleic acid
molecule encoding a chimeric protein are routine and well
known in the art (Sambrook et al., supra, 1989). For
example, the skilled artisan would recognize that a stop
codon in the 5' nucleic acid molecule must be removed and
that the two nucleic acid molecules must be linked such
that the reading frame of the 3' nucleic acid molecule is
preserved. Methods of transforming plants with nucleic
acid molecules also are well known in the art (see, for
example, Mohoney et al., U.S. patent number 5,463,174,
and Barry et al., U.S. patent number 5,463,17`=).
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As used herein, the term "operably linked,"
when used in reference to two nucleic acid molecules
" comprising a nucleic acid molecule encoding a chimeric
protein, means that the two nucleic acid molecules are
linked in frame such that a full-lengthchimeric protein
can be expressed. In particular, the 5' nucleic acid
molecule, which encodes the amino-terminal portion of the
chimeric protein, must be linked to the 3" nucleic acid
molecule, which encodes the carboxyl-terminal portion of
the chimeric protein, such that the carboxyl-terminal
portion of the chimeric protein is produced in the
correct reading frame.
The invention further provides a transgenic
angiosperm containing a nucleic acid molecule encoding a
chimeric protein, comprising a nucleic acid molecule
encoding a floral meristem identity gene product such as
AP1, CAL or LFY linked to a nucleic acid molecule
encoding a ligand binding domain. Such a transgenic
angiosperm is particularly useful because the angiosperm
can be induced to flower by contacting the angiosperm
with a ligand that binds the ligand binding domain.
Thus, the invention provides a method of promoting early
flowering or of converting shoot meristem to floral
meristem in a transgenic angiosperm containing a nucleic
25- acid molecule encoding a chimeric protein of the
invention, comprising expressing the nucleic acid
molecule encoding the chimeric protein in the angiosperm,
and contacting the angiosperm with a ligand that binds
the ligand binding domain, wherein binding of the ligand
to the ligand binding domain activates the floral
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meristem identity gene product. In particular, the
invention provides methods of promoting early flowering
or of converting shoot meristem to floral meristem in a
transgenic angiosperm containing a nucleic acid molecule
encoding a chimeric protein that consists of a nucleic
acid molecule encoding APi or CAL or LFY linked to a
nucleic acid molecule encoding a glucocorticoid receptor
ligand binding domain by contacting the transgenic
angiosperm with a glucocorticoid such as dexamethasone.
As used herein, the term "ligand" means a
naturally occurring or synthetic chemical or biological
molecule such as a simple or complex organic molecule, a
peptide, a protein or an oligonucleotide that
specifically binds a ligand binding domain. A ligand of
the invention can be used, alone, in solution or can be
used in conjunction with an acceptable carrier that can
serve to stabilize the ligand or promote absorption of
the ligand by an angiosperm.
One skilled in the art can readily determine
the optimum concentration of ligand needed to bind a
ligand binding domain and render a floral meristem
identity gene product active. Generally, a concentration
of about 1 nM to l M dexamethasone is useful for
activating floral meristem identity gene product activity
in a chimeric protein comprising a floral meristem
identity gene product and a glucocorticoid receptor
ligand binding domain (Lloyd et al., supra, 1994).
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A transgenic angiosperm expressing a chimeric
protein of the invention can be contacted with ligand in
a variety of manners including, for example, by spraying,
injecting or immersing the angiosperm. Further, a plant
may be contacted with a ligand by adding the ligand to
the plant's water supply or to the soil, whereby the
ligand is absorbed into the angiosperm.
The following examples are intended to
illustrate but not limit the present invention.
EXAMPLE I
Identification and characterization of the
Zea mays APETALA1 cDNA
This example describes the isolation and
characterization of the Zea mays ZAP-i "gene", which is
an ortholog of the Arabidopsis floral meristem identity
gene, AP1.
A. Identification and characterization of a nucleic acid
sequence encoding ZAP-I
The utility of using a cloned floral homeotic
gene from Arabidopsis to identify the putative ortholog
in maize has previously been demonstrated (Schmidt et
al., supra, (1993)).
As described in Mena et al. (Plant J.
8(6):845-854 (1995)), the maize ortholog of the
Arabidopsis AP1 floral meristem identity gene, was
isolated by screening a Zea mays ear cDNA library using
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the Arabidopsis AP1 cDNA (SEQ ID NO: 1) as a probe. A
cDNA library was prepared from wild-type immature ears as
described by Schmidt et al., supra, 1993, using an
Arabidopsis AP1 cDNA sequence as a probe. The
5 Arabidopsis AP1 cDNA (SEQ ID NO: 1), which is shown in
Figure 1 (SEQ ID NO 1), was used as the probe.
Low-stringency hybridizations with the AP1 probe were
conducted as described previously for the isolation of
ZAG1 using the AG cDNA as a probe (Schmidt et al., supra,
10 1993). Positive plaques were isolated and cDNAs were
recovered in Bluescript by in vivo excision.
Double-stranded sequencing was performed using the
Sequenase Version 2.0 kit (U.S. Biochemical, Cleveland,
Ohio) according to the manufacturer's protocol.
15 The cDNA sequence and deduced amino acid
sequence for ZAP1 are shown in Figure 4 (SEQ ID NOS: 7
and 8). The deduced amino acid sequence for ZAP1 shares
89o identity with Arabidopsis AP1 through the MADS domain
(amino acids 1 to 57) and 70% identity through the first
20 160 amino acids, which includes the K domain. The high
level of amino acid sequence identity between ZAP1 and
AP1 (SEQ ID NOS: 8 and 2), as well as the expression
pattern of ZAP1 in maize florets (see below), indicates
that ZAP1 is the maize ortholog of Arabidopsis AP1.
25 B, RNA expression pattern of ZAP1
Total RNA was isolated from different maize
tissues as described by Cone et al., Proc. Natl, Acad.
Sci., USA 83:9631-9635 (1986).
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RNA was prepared from ears or
tassels at early developing stages (approximately 2 cm in
size), husk leaves from developing ear shoots, shoots and
roots of germinated seedlings, leaves from 2 to 3 week
old plants and endosperm, and embryos at 18 days after
pollination. Mature floral organs were dissected from
ears at the time of silk emergence or from tassels at
several days pre-emergence. To study expression patterns
in the mature female flower, carpels were isolated and
the remaining sterile organs were pooled and analyzed
together. In the same way, stamens were dissected and
collected from male florets and the remaining organs
(excluding the glumes) were pooled as one sample.
RNA concentration and purity was determined by
absorbance at 260/280 nM, and equal amounts (10 /.cg) were
fractionated on formaldehyde-agarose gels. Gels were
stained in a solution of 0.125 g ml-1 acridine orange to
confirm the integrity of the RNA samples and the
uniformity of gel loading, then RNA was blotted on to
Hybond-W) membranes (Amersham International, Arlington
Heights, Illinois) according to the manufacturer's
instructions. Prehybridization and hybridization
solutions were prepared as previously described (Schmidt
et al., Science 238:960-963 (1987)).
The probe for ZAP1 RNA expression
studies was a 445 bp SacI-NsiI fragment from the 3' end
of the cDNA. Southern blot analyses were conducted to
establish conditions for specific hybridization of this
probe. No cross-hybridization was detected with
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hybridization at 60 C in 50o formamide and washes at 65 C
in 0.1x SSC and 0.5% SDS.
The strong sequence similarity between ZAP1 and
API indicated that ZAP1 was the ortholog of this
Arabidopsis floral meristem identity gene. As a first
approximation of whether the pattern of ZAP1 expression
paralleled that of AP1, a blot of total RNA from
vegetative and reproductive organs was hybridized with a
gene-specific fragment of the ZAP1 cDNA (nucleotides 370
to 820 of SEQ ID NO: 7). ZAP1 RNA was detected only in
male and female inflorescences and in the husk leaves
that surround the developing ear. No ZAP1 RNA expression
was detectable in RNA isolated from root, shoot, leaf,
endosperm, or embryo tissue. The restriction of ZAP1
expression to terminal and axillary inflorescences is
consistent with ZAP1 being the Arabidopsis AP1 ortholog.
Male and female florets were isolated from
mature inflorescences, and the reproductive organs were
separated from the remainder of the floret. RNA was
isolated from the reproductive and the sterile portions
of the florets. ZAP1 RNA expression was not detected in
maize stamens or carpels, whereas high levels of ZAP1
RNA were present in developing ear and tassel florets
from which the stamens and carpels had been removed.
Thus, the exclusion of ZAP1 expression in stamens and
carpels and its inclusion in the RNA of the
non-reproductive portions of the floret (lodicules, lemma
and palea) is similar to the pattern of expression of API
in flowers of Arabidopsis.
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EXAMPLE II
Conversion of shoot meristem to floral meristem in an
APETALA1 transgenic plant
This example describes methods for producing a
transgenic Arabidopsis plant, in which shoot meristem is
converted to floral meristem.
A. Ectopic expression of APETALA1 converts inflorescence
shoots into flowers
Transgenic plants that constitutively express
AP1 from the cauliflower mosaic virus 35S (CaMV35S)
promoter were produced to determine whether ectopic AP1
expression could convert shoot meristem to floral
meristem. The AP1 coding sequence was placed under
control of the cauliflower mosaic virus 35S promoter
(Odell et al., supra, 1985) as follows. BamHI linkers
were ligated to the HincIl site of the full-length AP1
complementary DNA (Mandel et al., supra, (1992))
in pAM116, and the
resulting BamHI fragment was fused to the cauliflower
mosaic virus 35S promoter (Jack et al., Cell 76:703-716
(1994)) in pCGN18 to create pAM563.
Transgenic AP1 Arabidopsis plants of the
Columbia ecotype were generated by selecting
kanamycin-resistant plants after Agrobacteriurn-mediated
plant transformation using the in planta method (Bechtold
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et al., C.R. Acad. Sci. Paris 316:1194-1199 (1993)).
. All analyses were
performed in subsequent generations. Approximately 120
independent transgenic lines that displayed the described
phenotypes were obtained.
Remarkably, in 35S-AP1 transgenic plants, the
normally indeterminate shoot apex ) prematurely
terminated as a floral meristem and formed a terminal
flower. In addition, all lateral meristems that normally
would produce inflorescence shoots also were converted
into solitary flowers. These results demonstrate that
ectopic expression of AP1 in shoot meristem is sufficient
to convert shoot meristem to floral meristem, even though
AP1 normally is not absolutely required to specify floral
meristem identity.
B LEAFY is not required for the conversion of
inflorescence shoots to flowers in an APETALAI
transcrenic -plant
To determine whether the 35S-AP1 transgene
causes ectopic LFY activity, and whether ectopic LFY
activity is required for the conversion of shoot meristem
to floral meristem, the 35S-AP1 transgene was introduced
into Arabidopsis 1fy mutants. The 35S-AP1 transgene was
crossed into the strong lfy-6 mutant background and the F,
progeny were analyzed.
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Lfy mutant plants containing the 35S-API
transgene displayed the same conversion of apical and
lateral shoot meristem to floral meristem as was observed
in transgenics containing wild type LFY. However, the
5 resulting flowers had the typical lfy mutant phenotype,
in which floral organs developed as sepaloid and
carpelloid structures, with an absence of petals and
stamens. These results demonstrate that LFY is not
required for the conversion of shoot meristem to floral
10 meristem in a transgenic angiosperm that ectopically
expresses APl.
C. APETALAI is not sufficient to specify organ fate
As well as being involved in the early step of
specifying floral meristem identity, API also is involved
15 in specifying sepal and petal identity at a later stage
in flower development. Although API RNA is initially
expressed throughout the young flower primordium, it is
later excluded from stamen and carpel primordia (Mandel
et al., Nature 360:273-277 (1992)). Since the
20 cauliflower mosaic virus 35S promoter is active in all
floral organs, 35S-API transgenic plants are likely to
ectopically express AP1 in stamens and carpels. However,
35S-API transgenic plants had normal stamens and carpels,
indicating that AP1 is not sufficient to specify sepal
25 and petal organ fate.
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D, Ectopic expression of APETALA1 causes early flowering
In addition to its ability to alter
inflorescence meristem identity, ectopic expression of
API also influences the vegetative phase of plant growth.
Wild-type plants have a vegetative phase during which a
basal rosette of leaves is produced, followed by the
transition to reproductive growth. The transition from
vegetative to reproductive growth was measured both in
terms of the number of days post-germination until the
first visible flowers were observed, and by counting the
number of leaves. Under continuous light, wild-type and
35S-AP1 transgenic plants flowered after producing 9.88
1.45 and 4.16 0.97 leaves, respectively. Under short-day
growth conditions (8 hours light, 16 hours dark, 24 C),
wild-type and 35S-AP1 transgenic plants flowered after
producing 52.42 3.47 and 7.4 1.18 leaves, respectively.
In summary, under continuous light growth
conditions, flowers appear on wild-type Arabidopsis
plants after approximately 18 days, whereas the 35S-AP1
transgenic plants flowered after an average of only 10
days. Furthermore, under short-day growth conditions,
flowering is delayed in wild-type plants until_
approximately 10 weeks after germination, whereas, 35S-
AP1 transgenic plants flowered in less than 3 weeks.
Thus, ectopic AP1 activity significantly reduced the time
to flowering and reduced the delay of flowering caused by
short day growth conditions.
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EXAMPLE III
zsolation and characterization of the Arabidopsis and
Brassica oleracea CAULIFLOWER aenes
' This example describes methods for isolating
and characterizing the Arabidopsis and Brassica oleracea
CAL genes.
A. Isolation of the ArabidoDsis and Brassica oleracea
[, AUL TFLOWER crenes
Genetic evidence that CAL and APl proteins may
be functionallyrelated indicated that these proteins may
share similar DNA sequences. In addition, DNA blot
hybridization revealed that the Arabidopsis genome
contains a gene that is closely related to APi. The CAL
gene, which is closely related to APi, was isolated and
identified as a member of the family of Arabidopsis MADS
domain genes known as the AGAMOUS-like (AGL) genes.
Hybridization with an AP1 probe was used to
isolate a 4.8-kb Eco RI genomic fragment of CAL. The
corresponding CAL complementary DNA (pBS85) was cloned by
reverse transcription-polymerase chain reaction (RT-PCR)
with the oligonucleotides AGLIO-1
(5'-GATCGTCGTTATCTCTCTTG-3'; SEQ ID NO: 25) and AGL10-12
(5'-GTAGTCTATTCAAGCGGCG-3'; SEQ ID NO: 26).
The Arabidopsis CAL cDNA encodes a putative 255
amino acid protein (Figure 5; SEQ ID NO: 10) having a
calculated molecular weight of 30.1 kD and an isoelectric
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point of 8.78. The deduced amino acid sequence for CAL
contains a MADS domain which generally is present in a
class of transcription factors. The MADS domains of CAL
and AP1 were markedly similar, differing in only 5 of 56
amino acid residues, 4 of which represent conservative
replacements. Overall, the putative CAL protein is 76%
identical to AP1; with allowance for conservative amino
acid substitutions, the two proteins are 88o similar.
These results indicate that CAL and APl may recognize
similar target sequences and regulate many of the same
genes involved in floral meristems identity.
CAL was mapped to the approximate location of
the loci identified by classical genetic means for the
cauliflower phenotype (Bowman et al., Development 119:721
(1993)).
Restriction fragment length polymorphism (RFLP) mapping
filters were scored and the results analyzed with the
Macintosh version of the Mapmaker program as described by
Rieter et al., (Proc. Natl. Acad. Sci., USA, 89:1477
(1992)). The
results localized CAL to the upper arm of chromosome 1,
near marker X235.
A genomic fragment spanning the CAL gene was
used to transform cal-1 apl-i plants. A 5850-bp Bam HI
fragment containing the entire coding region of the
Arabidopsis CAL gene as well as 1860 bp upstream of the
putative translational start site was inserted into the
pBIN19 plant transformation vector (Clontech, Palo Alto,
California) and used for transformation of root tissue
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from caI-I api-i plants as described by Valvekens et al.
(Proc. Natl. Acad. Sci., USA 85:5536 (1988))-
Seeds were harvested
from primary transformants, and all phenotypic analyses
were performed in subsequent generations. Four
independent lines transformed with CAL showed a
complementation of the cauliflower (cal) phenotype and
displayed a range of phenotypes similar to those
exhibited by api mutants. These results demonstrated
that CAL functions to convert shoot meristem to floral
meristem.
In order to identify regions of functional
importance in the CAL protein, cal mutants were generated
and analyzed. The cal alleles were isolated by
mutagenizing seeds homozygous for the api-I allele in Ler
with 0.1% or 0.05% ethylmethane sulfonate (EMS) for 16
hours. Putative new cal alleles were crossed to ca1-I
api-i chlorina plants to verify allelism. Two sets of
oligonucleotides were used to amplify and clone new
alleles: oligos AGL10-1 (SEQ ID NO: 25) and AGL10-2
(5'-GATGGAGACCATTAAACAT-3; SEQ ID NO: 27) for the 5'
portion and oligos AGL10-3 (5'-GGAGAAGGTACTAGAACG-3'; SEQ
ID NO: 28) and AGL10-4 (5'-GCCCTCTTCCATAGATCC-3'; SEQ ID
NO: 29) for the 3' portion of the gene. All coding
reaions and intron-exon boundaries of the mutant alieles
were sequenced.
Sequence analysis of the cal-i allele, which
exists in the wild-type Wassilewskija (WS) ectoype,
revealed a cluster of three amino acid differences in the
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seventh exon, relative to the wild-type gene product from
Landsberg erecta (Ler) (Figure 8). One or more of these
amino acid differences can be responsible for the cal phenotype, because the
cal-i gene was expressed normally
5 and the transcribed RNA was correctly spliced in the WS
background. The three additional cal alleles that were
isolated, designated caZ-2, caI-3, and cal-4, exhibited
phenotypes similar to that of the cal-i allele.
Sequence analyses revealed a single missense
10 mutation for each (Figure 8). Since mutations in the
cal-2 and cal-3 alleles lie in the MADS domain, these
mutations can affect the ability of CAL to bind DNA and
activate its target genes. Because the cal-4 allele
contains a substitution in the K domain, a motif thought
15 to be involved in protein-protein interactions, this
mutation can affect the ability of CAL to form homodimers
or to interact with other proteins such as AP1.
B. RNA expression pattern of CAULIFLOWER
To characterize the temporal and spatial
20 pattern of CAL RNA accumulation, RNA in situ
hybridizations were performed using a CAL-specific probe.
35S-labeled antisense CAL and BoCAL mRNA was synthesized
from Sca 1-digested cDNA templates and hybridized to 8,um
sections of Arabidopsis Ler or Brassica oleracea
25 inflorescences. The probes did not contain any MADS box
sequences in order to avoid cross-hybridization with
other MADS box genes. Hybridization conditions were as
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previously described (Drews et al., Cell 65:991 (1991)).
As with AP1, CAL RNA accumulated in young
flower primordia, consistent with the ability of CAL to
substitute for APi in specifying floral meristems. In
contrast to AP1 RNA, however, which accumulated at high
levels throughout sepal and petal development, CAL RNA
was detected only at very low levels in these organs.
These results demonstrate that CAL was unable to
substitute for AP1 in specifying sepals and petals, at
least in part as a result of the relatively low levels of
CAL RNA in these developing organs.
C. Molecular Basis of the cauliflower ghenotyge
The cal phenotype in Arabidopsis is similar to
the inflorescence structure that develops in the closely
related species Brassica oleracea var. botrytis, the
cultivated garden variety of cauliflower, indicating that
the CAL gene can contribute to the cal phenotype of this
agriculturally important species. Thus, CAL gene
homologs were isolated from a Brassica oleracea line that
produces wild-type flowers (BoCAL) and from the common
garden variety of cauliflower Brassica oleracea var.
bo tryti s (BobCAL) .
The single-copy BobCAL gene (Snowball Y
Improved, NK Lawn & Garden, Minneapolis, MN) was isolated
from a size-selected genomic library in XBlueStar
(Novagen) on a 16-kbp BamHI fragment with the Arabidopsis
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CAL gene as a probe. The BoCAL gene was isolated from a.
rapid cycling line (Williams and Hill, Science 232:1385
(1986)) by PCR on both RNA and genomic DNA. The cDNA was
isolated by RT-PCR using the oligonucleotides: Bobl
5(5'-TCTACGAGAAATGGGAAGG-3'; SEQ ID NO: 30) and Bob2
(5'-GTCGATATATGGCGAGTCC-3'; SEQ ID NO: 31). The 5'
portion of the gene was obtained using oligonucleotides
Bob 1 (SEQ ID NO: 30) and Bob4B
(5'-CCATTGACCAGTTCGTTTG-3'; SEQ ID NO: 32). The 3'
portion was obtained using oligonucleotides Bob3
(5'-GCTCCAGACTCTCACGTC-3'; SEQ ID NO: 33) and Bob2 (SEQ
ID NO: 31).
RNA in situ hybridizations were performed to
determine the expression pattern of BoCAL gene from
Brassica oleracea. As in Arabidopsis, BoCAL RNA
accumulated uniformly in early floral primordia and later
was excluded from the cells that give rise to stamens and
carpels.
DNA sequence analyses revealed that the open
reading frame of the BoCAL gene is intact, whereas that
of the BobCAL gene is interrupted by a stop codon in
exon 5(Figure 8). Translation of the resulting BobCAL
protein product is truncated after only 150 of the
wild-type 255 amino acids. Because similar stop codon
mutations in the fifth exon of the Arab%dopsis AP1 coding
sequence result in plants having a severe api phenotype,
the BobCAL protein likely is not functional. These
results indicate that, as in Arabidopsis, the molecular
basis for the cauliflower phenotype in Brassica oleracea
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var. botrytis is due, at least in part, to a mutation in
the BobCAL gene.
EXAMPLE IV
Conversion of inflorescenc shoots into flowers in an
CAULIFLOWER transgenic 81ant
This example describes methods for producing a
transgenic CAL plant.
A Ectopic expression of CAULTF WER converts
inflorescence shoots to flowers
Transgenic Arabidopsis plants that ectopically_
express CAL in shoot meristem were generated. The
full-length CAL cDNA was inserted downstream of the 35S
cauliflower mosaic virus promoter in the EcoRI of pMON530
(Monsanto Co. Co., St. Louis, Missouri) This plasmid was
introduced into Agrobacterium strain ASE (check) and used
to transform the Columbia ecotype of Arabidopsis using a
modified vacuum infiltration method described by Bechtold
et al. (supra, 1993). The 96 lines generated that
harbored the 35S-CAL construct had a range of weak to
strong phenotypes. The transgenic plants with the
strongest phenotypes (27 lines) closely resembled the tfl
mutant.
35S-CAL transgenic plants had converted apical
and lateral inflorescence shoots into flowers and showed
an early flowering phenotype. These results demonstrate
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that CAL is sufficient for the conversion of shoots to
flowers and for promoting early flowering.
EXAMPLE V
Conversion of shoots into flowers in a
LEAFY transgenic plant
This example describes methods for producing a
transgenic LFY Arabidopsis and aspen.
A. Conversion of Arabidozasis shoots by LEAFY
Transgenic Arabidopsis plants were generated by
transforming Arabidopsis with LFY under the control of
the cauliflower mosaic virus 35S promoter (CaMV35S)(Odell
et al., supra, (1985)). A LFY complementary cDNA (Weigel
et al, Cell 69:843-859 (1992))
was inserted into a T-DNA
transformation vector containing a CaMV 35S promoter/3'
nos cassette (Jack et al., supra, 1994). Transformed
seedlings were selected for kanamycin resistance.
Several hundred transformants in three different genetic
backgrounds (Nossen, Wassilewskija and Columbia) were
recovered and several lines were characterized in detail.
High levels of LFY RNA expression were detected
by northern blot analysis. In general, Nossen lines had
weaker phenotypes, especially when grown in short days.
The 35S-LFY transgene of line DW151.117 (ecotype
Wassilewskija) was introgressed into the erecta
background by backcrossing to a Landsberg erecta strain.
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Plants were grown under 16 hours light and 8 hours dark.
The 35S-LFY transgene provided at least as much LFY
activity as the endogenous gene and completely suppressed
the 3fy mutant phenotype when crossedinto the background
5 of the lfy-6 null allele.
Most 35S-LFY transgenic plants lines
demonstrated a very similar, dominant and he'ritable
phenotype. Secondary shoots that arose in lateral
positions were consistently replaced by solitary flowers,
10 and higher-order shoots were absent. Although the number
of rosette leaves was unchanged from the wild type,
35S-LFY plants flowered earlier than wild type; the
solitary flowers in the axils of the rosette leaves
developed and opened precociously. In addition, the
15 primary shoot terminated with a flower. In the most
extreme cases, a terminal flower was formed immediately
above the rosette. This gain of function phenotype
(conversion of shoots to flowers) is the opposite of the
Ify loss of function phenotype (conversion of flowers to
20 shoots). These results demonstrate that LFY encodes a
developmental switch that is both sufficient and
necessary to convert shoot meristem to flower meristem.
The effects of constitutive LFY expression
differ for primary and secondary shoot meristems.
25 Secondary meristems were transformed into flower
meristem, apparently as soon as it developed, and
produced only a single, solitary flower. In contrast,
primary shoot meristem produced leaves and lateral
flowers before being consumed in the formation of a
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terminal flower. These developmental differences
indicate that a meristem must acquire competence to
respond to the activity of a floral meristem identity
gene such as LFY.
B. Conversion of aspen shoots by LEAFY
Given that constitutive expression of LFY
induced precocious flowering during the vegetative phase
of Arabidopsis, the effect of LFY on the flowering of
other species was examined. The perennial tree, hybrid
aspen, is derived from parental species that flower
naturally only after 8-20 years of growth (Schopmeyer
(ed.), USDA Aariculture Handbook 450: Seeds of Woody
Plants in the United States, Washington DC, USA: US
Government Printing Office, pp. 645-655 (1974)). 35S-LFY
aspen plants were obtained by Agrobacterium-mediated
transformation of stem segments and subsequent
regeneration of transgenic shoots in tissue culture.
Hybrid aspen was transformed exactly as
described by Nilsson et al. (Transcren. Res. 1:209-220
(1992)).
Levels of LFY RNA expression were similar to those of
35S-LFY Arabidopsis, as determined by northern blot
analysis. The number of vegetative leaves varied between
different regenerating shoots, and those with a higher
number of vegetative leaves formed roots, allowing for
transfer to the greenhouse. Individual flowers were
removed either from primary transformants that had been
transferred to the greenhouse, or from catkins collected
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in spring, 1995, at Carlshem, ITmea, Sweden) from a tree
whose age was determined by counting the number of annual
rings in a core extracted with an increment borer at 1.5
meters above ground level. Flowers were fixed in
formaldehyde/acetic acid/ethanol and destained in ethanol
before photography.
The overall phenotype of 35S-LFY aspen was
similar to that of 35S-LFY Arabidopsis. In wild-type
plants of both species, flowers normally are formed in
lateral positions on inflorescence shoots. In aspen,
these inflorescence shoots, called catkins, arise from
the leaf axils of adult trees. In both 35S-LFY
Arabidopsis and 35S-LFY aspen, solitary flowers were
formed instead of shoots in the axils of vegetative
leaves. Moreover, as in Arabidopsis, the secondary
shoots of trangenic aspen were more severely affected
than the primary shoot.
Regenerating 35S-LFY aspen shoots initially
produced solitary flowers in the axils of normal leaves.
However, the number of vegetative leaves was limited, and
the shoot meristem was prematurely consumed in the
formation of an aberrant terminal flower. Precocious
flower development was specific to 35S-LFY transformants
and was not observed in non-transgenic controls.
Furthermore, not a single instance of precocious flower
development has been observed in more than 1,500 other
lines of transgenic aspen generated with various
constructs from 1989 to 1995 at the Swedish University of
Agricultural Sciences. These results demonstrate that a
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heterologous floral meristem identity gene product is
active in an angiosperm.
Although the invention has been described with
reference to the examples above, it should be understood
that various modifications can be made without departing
from the spirit pf the invention. Accordingly, the
invention is limited only by the following claims.
