AU725704B2

AU725704B2 - Maize and cauliflower apetala1 gene products and nucleic acid molecules encoding same

Info

Publication number: AU725704B2
Application number: AU61600/96A
Authority: AU
Inventors: Martin F. Yanofsky
Original assignee: University of California
Current assignee: University of California
Priority date: 1996-06-05
Filing date: 1996-06-05
Publication date: 2000-10-19
Anticipated expiration: 2016-06-05
Also published as: WO1997046078A1; AU6160096A

Description

WO 97/46078 PCTUS96/09453 MAIZE AND CAULIFLOWER APETALAI GENE PRODUCTS AND NUCLEIC ACID MOLECULES ENCODING SAME This work was supported by grant DCB-9018749 awarded by the National Science Foundation and by grant USDA 93-37304 awarded by the United States Department of Agriculture. The United States Government has certain rights in this invention.

BACKGROUND OF THE INVENTION FIELD OF THE INVENTION This invention relates generally to the field of plant genetic engineering and more specifically to genes involved in the regulation of plant reproductive development.

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, rice and hops is dependent upon flowering.

Early in the life cycle of a flowering plant, 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.

WO 97/46078 PCT/US96/09453 2 However, the factors responsible for the transition from vegetative to reproductive growth, and the onset of flowering, are poorly understood.

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 WO 97/46078 PCTIUS96/09453 3 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 be reproductively mature before selective breeding can proceed.

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 decades, even if only a few rounds of selection were performed.

Methods of promoting early reproductive development can make breeding of long generation seed plants such as trees practical for the first time.

Methods of promoting early reproductive development 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 reproductive development in a seed plant have not yet been described. Thus, there is a need for methods that promote early reproductive development. The present invention satisfies this need and provides related advantages as well.

4 SUMMARY OF THE INVENTION According to the a first aspect of the present invention there is provided a substantially purified nucleic acid molecule encoding an API gene product having an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8.

According to a second aspect of the present invention there is provided a substantially purified nucleic acid molecule having a nucleic acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7.

A method of producing an API gene product, comprising expressing nucleic acid molecule as described above also forms a part of the invention, as does a vector comprising an nucleic acid molecule encoding an API gene product as described above.

According to a third aspect of the present invention there is provided a substantially purified API :gene product having an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8.

In addition, the invention provides an antibody, such as a monoclonal antibody, that specifically binds the API gene product described above.

•g.

According to a fourth aspect of the present invention there is provided an expression vector, comprising a nucleic acid molecule encoding an API gene product having an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO:6 and SEQ ID NO: 8.

Typically said expression vector is a plant 5 expression vector comprising a heterologous regulatory element, preferably a constitutive regulatory element such as cauliflower mosac virus 35S promotor but it may also be an inducable regulatory element.

In addition, the present invention provides a kit for converting shoot meristem to floral meristem in an angiosperm, comprising the plant expression vector describe above. The invention additionally provides a kit for promoting early reproductive development in a seed plant, comprising the plant expression vector described above.

Throughout the description and claims, except where the context requires otherwise, due to express language or necessary implication, the words "comprising", "comprises", or "comprise" are used in the sense of "including", that is the features specified may be associated with further features in various embodiments of 20 the ivention.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 illustrates a western-blot analysis of tissues from wild type and mutant Arabidopsis plants with anti-AP1 antisera.

DETAILED DESCRIPTION OF THE INVENTION The invention relates generally to a nonnaturally occurring seed plant containing a first ectopically expressible nucleic acid molecule encoding a first floral meristem identity gene product, provided that the nucleic acid molecule is not ectopically expressed due to a mutation in an endogenous TERMINAL FLOWER gene. For example, the transgenic seed plant may contain a first ectopically expressible floral meristem identity gene product such as APETALA1 (AP1), CAULIFLOWER (CAL) or LEAFY 5a ectopically expressible floral meristem identity gene product such as APETALAl (AP1), CAULIFLOWER (CAL) or LEAFY (LFY). A transgenic seed plant can be, for example, an angiosperm such as a cereal plant, leguminous plant, oilseed plant, hardwood tree, fruit-bearing plant or ornamental flower or a gymnosperm such as a coniferous tree.

S

WO 97/46078 PCT/US96/09453 6 A flower, like a leaf or shoot, is derived from the shoot apical meristem, which is a collection of undifferentiated cells set aside during embryogenesis.

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 develops later. The transition from vegetative to reproductive development is the consequence of a process termed floral induction (Yanofsky, Ann. Rev. Plant Physiol. Plant Mol. Biol. 46:167-188 (1995), which is incorporated herein by reference).

Once induced, shoot apical meristem either 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.

Floral meristem differentiates 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 WO 97/46078 PCTIUS96/09453 7 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, APETALAI), 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 (apl), 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 an angiosperm. In addition, floral organ identity genes encode proteins that determine whether sepals, petals, stamens or carpels are formed during floral development (Yanofsky, supra, 1995; Weigel, Ann. Rev. Genetics 29:19-39 (1995), which is incorporated herein by reference). Some of the floral meristem identity gene products also have a role in specifying floral organ identity.

Floral meristem identity genes have been identified by characterizing genetic mutations that WO 97/46078 PCT/US96/09453 8 prevent or alter floral meristem formation. Among floral meristem identity gene mutations in Arabidopsis thaliana, 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 later-arising (apical) flowers. In comparison, mutations in the floral meristem identity gene APETALA1 (API) result in replacement of a few basal flowers by inflorescence shoots that are not subtended by leaves.

An apical flower produced in an apl 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 Ify apl 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 API genes, a third locus that greatly enhances the apl mutant phenotype has been identified in Arabidopsis. This locus, designated CAULIFLOWER (CAL), derives its name from the resulting "cauliflower" phenotype, which is strikingly similar to the common garden variety of cauliflower (Kempin et al., Science 267:522-525 (1995), which is incorporated herein by reference). In an apl cal double mutant, floral meristem 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, i* WO 97/46078 PCT/US96/09453 9 an Arabidopsis mutant lacking only CAL, such as 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 apl mutant behaves as shoot meristem in an apl cal double mutant, CAL can largely substitute for API in specifying floral meristem. These genetic data indicate that CAL and API encode activities 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 (flowers with shoot-like characteristics; Bowman et al., Development 119:721-743 (1993), which is incorporated herein by reference). Also, mutations in the CLAVATAI (CLV) gene result in an enlarged meristem and lead to a variety of phenotypes (Clark et al., Development 119:397-418 (1993)). In a clvl apl 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.

WO 97/46078 PCT/US96/09453 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, tfl 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 meristem. TFL also acts directly or indirectly to negatively regulate AP1 and LFY expression in shoot meristem since these API and LFY are ectopically expressed in the shoot meristem of tfl 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 seed plant of the invention. Such tfl mutants, however, which have a mutation in an endogenous TERMINAL FLOWER gene, 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 in an angiosperm. 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 in an angiosperm. Thus, the present invention provides a non-naturally occurring seed plant such as an angiosperm or gymnosperm that contains a first ectopically WO 97/46078 PCT/US96/09453 11 expressible nucleic acid molecule encoding a first 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 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 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 negative regulation by TFL.

The term "ectopically expressible" is used herein to refer to a nucleic acid molecule encoding a floral meristem identity gene product that can be expressed in a tissue other than a tissue in which it normally is expressed or at a time other than the time at which it normally is expressed, provided that the floral meristem identity gene product is not expressed from its native, naturally occurring promoter. Ectopic expression of a floral meristem identity gene product is a result of the expression of the gene coding region from a heterologous promoter or from a modified variant of its WO 97/46078 PCT/US96/09453 12 own promoter, such that expression of the floral meristem identity gene product is no longer in the tissue in which it normally is expressed or at the time at which it normally is expressed. An exogenous nucleic acid molecule encoding an AP1 gene product under control of its native, wild type promoter, for example, does not constitute an ectopically expressible nucleic acid molecule encoding a floral meristem identity gene product. However, a nucleic acid molecule encoding an AP1 gene product under control of a constitutive promoter, which results in expression of API in a tissue such as shoot meristem where it is not normally expressed, is an ectopically expressible nucleic acid molecule as defined herein.

Actual ectopic expression of a floral meristem identity gene is dependent on various factors and can be constitutive or inducible expression. For example, AP1, which normally is expressed in floral meristem, is ectopically expressible in the shoot meristem of an angiosperm. As disclosed herein, when a floral meristem identity gene product such as AP1, CAL or LFY is ectopically expressed in shoot meristem in an angiosperm, the shoot meristem is converted to floral meristem and early reproductive development can occur (see Examples I, III and IV).

An ectopically expressible nucleic acid molecule encoding a floral meristem identity gene product can be expressed prior to the time in development at which the corresponding endogenous gene normally is WO 97/46078 PCT/US96/09453 13 expressed. For example, an Arabidopsis plant grown under continuous light conditions expresses API just prior to day 18, when normal reproductive development (flowering) begins. However, as disclosed herein, API can be ectopically expressed in shoot meristem prior to day 18, resulting in early conversion of shoot meristem to floral meristem and early reproductive development. As disclosed in Example ID, a transgenic Arabidopsis plant that ectopically expresses API in shoot meristem under control of a constitutive promoter can flower at day which is earlier than the time of reproductive development for a non-transgenic plant grown under the same conditions (day 18). It is recognized that in some transgenic seed plants containing, for example, an exogenous nucleic acid molecule encoding AP1 under control of a constitutive promoter, neither the exogenous nor endogenous AP1 will be expressed. Such transgenic plants in which AP1 gene expression is cosuppressed, although not characterized by early reproductive development, also can be valuable as disclosed below.

As used herein, the term "floral meristem identity gene product" means a gene product that promotes conversion of shoot meristem to floral meristem in an angiosperm. As disclosed herein in Examples I, II and III, expression of a floral meristem identity gene product such as AP1, CAL or LFY in shoot meristem can convert shoot meristem to floral meristem in an angiosperm. Furthermore, ectopic expression of a floral meristem identity gene product also can promote early reproductive development (see Example ID).

WO 97/46078 PCTIUS96/09453 14 A floral meristem identity gene product is distinguishable from a late flowering gene product or an early flowering gene product. The use of a late flowering gene product or an early flowering gene product is not encompassed within the scope of the present invention. In addition, reference is made herein to an "inactive" floral meristem identity gene product, as exemplified by the product of the Brassica oleracea var.

botrytis CAL gene (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. An inactive floral meristem identity gene product such as BobCAL is excluded from the meaning of the term "floral meristem identity gene product" as defined herein.

A floral meristem identity gene product can be, for example, an AP1 gene product having the amino acid sequence of SEQ ID NO: 2, which is a 256 amino acid gene product encoded by the Arabidopsis thaliana API cDNA.

The Arabidopsis API cDNA encodes a highly conserved MADS domain, which can function as a DNA-binding domain, and a K domain, which has structural similarity to the coiled-coil domain of keratins and can be involved in protein-protein interactions.

As used herein, the term "APETALA1," "API" or "AP1 gene product" 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 amino acid identity with the amino acid sequence of SEQ WO 97/46078 PCTIUS96/09453 ID NO: 2 in the region from amino acid 1 to amino acid 163 or with the amino acid sequence of SEQ ID NO: 8 in the region from amino acid 1 to amino acid 163. Like other floral meristem identity gene products, AP1 promotes conversion of shoot meristem to floral meristem in an angiosperm. An API gene product useful in the invention can be, for example, Arabidopsis AP1 having the amino acid sequence of SEQ ID NO: 2; Brassica oleracea AP1 having the amino acid sequence of SEQ ID NO: 4; Brassica oleracea var. botrytis AP1 having the amino acid sequence of SEQ ID NO: 6 or Zea mays AP1 having the amino acid sequence of SEQ ID NO: 8.

In wild-type Arabidopsis, API 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 API increases as the floral meristem increases in size; no API expression is detectable in shoot meristem. In later stages of development, API expression ceases in cells that will give rise to reproductive organs of a flower (stamens and carpels), but is maintained in cells that will give rise to non-reproductive organs (sepals and petals; Mandel, supra, 1992). Thus, in nature, API expression is restricted to floral meristem and to certain regions of the flowers that develop from this meristemic tissue.

WO 97/46078 PCT/US96/09453 16 CAULIFLOWER (CAL) is another example of a floral meristem identity gene product. As used herein, the term "CAULIFLOWER," "CAL" or "CAL gene product" 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 amino acid identity with the amino acid sequence of SEQ ID NO: 10 in the region from amino acid 1 to amino acid 160 or with the amino acid sequence of SEQ ID NO: 12 in the region from amino acid 1 to amino acid 160.

A CAL gene product is exemplified by the Arabidopsis CAL gene product, which has the amino acid sequence of SEQ ID NO: 10, or the Brassica oleracea CAL gene product, which has the amino acid sequence of SEQ ID NO: 12. As disclosed herein, CAL, like AP1, contains a MADS domain and a K domain. The MADS domains of CAL and AP1 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 AP1 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 API, 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. Thus, in nature, CAL is expressed in floral meristem and, to a lesser extent, in the organs of developed flowers.

WO 97/46078 PCT/US96/09453 17 The skilled artisan will recognize that, due to the high sequence conservation between AP1 and CAL, a novel ortholog can be categorized as both a CAL and an AP1, as defined herein. However, if desired, an AP1 ortholog can be distinguished from a CAL ortholog by demonstrating a greater similarity to Arabidopsis AP1 than to any other MADS box gene, such as CAL, as set forth in Purugganan et al. (Genetics 140:345-356 (1995), which is incorporated herein by reference). Furthermore, AP1 can be distinguished from CAL by its ability to complement, or restore a wild-type phenotype, when introduced into a strong apl mutant. For example, introduction of Arabidopsis API (AGL7) complements the phenotype of the strong apl-i mutant; however, introduction of CAL (AGLIO) into a cal-1 apl-1 mutant plant yields the apl-i single mutant phenotype, demonstrating that CAL cannot complement the apl-1 mutation (see, for example, Mandel et al., supra, 1992; Kempin et al., supra, 1995). Thus, AP1 can be distinguished from CAL, if desired, by the ability of a nucleic acid molecule encoding AP1 to complement a strong apl mutant such as apl-1 or LEAFY (LFY) is yet another example of a floral meristem identity gene product. As used herein, the term "LEAFY" or "LFY" or "LFY gene product" 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 amino acid identity with the amino acid sequence of SEQ ID NO: 16. In nature, LFY is expressed in floral meristem as well as during vegetative WO 97/46078 PCT/US96/09453 18 development. As disclosed herein, ectopic expression in shoot meristem of a floral meristem identity gene product, which normally is expressed in floral meristem, can convert shoot meristem to floral meristem in an angiosperm. Under appropriate conditions, ectopic expression in shoot meristem of a floral meristem identity gene product such as AP1, CAL, LFY, or a combination thereof, can promote early reproductive development.

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 angiosperm species, and these gene orthologs show remarkably similar patterns of RNA expression. Mutations in gene orthologs 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 (1993); and Veit et al., Plant Cell 5:1205-1215 (1993), each of which is incorporated herein by reference). 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), which WO 97/46078 PCT/US96/09453 19 is incorporated herein by reference) 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.

Floral meristem identity genes in particular are conserved among distantly related angiosperm and gymnosperm species. For example, a gene ortholog of Arabidopsis API has been isolated from Antirrhinum majus (snapdragon; Huijser et al., EMBO J. 11:1239-1249 (1992), which is incorporated herein by reference). As disclosed herein, an ortholog of Arabidopsis API also has been isolated from Brassica oleracea var. botrytis (cauliflower) and Zea Mays (maize; see Example VA).

Furthermore, API orthlogs also can be isolated from gymnosperms. Similarly, gene orthologs of Arabidopsis LFY have been isolated from angiosperms such as Antirrhinum majus, tobacco and poplar tree and from gymnosperms such as Douglas fir (Coen et al., Cell, 63:1311-1322 (1990); Kelly et al., Plant Cell 7:225-234 (1995); and Rottmann et al., Cell Biochem. Suppl. 17B: 23 (1993); Strauss et al., Molec. Breed 1:5-26 (1995), each of which is incorporated herein by reference). The conservation of floral meristem identity gene products in non-flowering plants such as coniferous trees indicates that floral meristem identity genes can promote the reproductive development of gymnosperms as well as angiosperms.

WO 97/46078 PCT/US96/09453 The characterization of apl and Ify mutants also indicates that floral meristem identity gene products such as AP1 and LFY function similarly in distantly related plant species. For example, a mutation in the Antirrhinum API ortholog results in a phenotype similar to the Arabidopsis apl indeterminate flower within a flower phenotype (Huijser et al., supra, 1992).

In addition, a mutation in the Antirrhinum LFY ortholog results in a phenotype similar to the Arabidopsis Ify mutant phenotype (Coen et al., supra, 1995) A floral meristem identity gene product also can function across species boundaries. For example, introduction of a nucleic acid molecule encoding Arabidopsis LFY into a heterologous seed plant such as tobacco or aspen results in early reproductive development (Weigel and Nilsson, Nature 377:495-500 (1995), which is incorporated herein by reference). As disclosed herein, 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 a heterologous seed plant such as corn, wheat, rice or pine and, upon ectopic expression, can promote early reproductive development in the transgenic seed plant. Furthermore, as disclosed herein, the conserved nature of the API, CAL and LFY coding sequences among diverse seed plant species allows a nucleic acid molecule encoding a floral meristem identity gene product isolated from essentially any seed plant to be introduced into essentially any other seed plant, wherein, upon appropriate expression of the introduced nucleic acid WO 97. J78 ICT/US96/09453 21 molecule in the seed plant, the floral meristem identity gene product promotes early reproductive development in the seed plant.

If desired, a novel API, CAL or LFY coding sequence can be isolated from a seed plant using a nucleotide sequence as a probe and methods well known in the art of molecular biology (Sambrook et al. (eds.), Molecular Clonin: A Laborator Manual (Second Edition), Plainview, NY: Cold Spring Harbor Laboratory Press (1989), which is incorporated herein by reference). As exemplified herein and discussed in detail below (see Example VA), an API ortholog from Zea Mays (maize; SEQ ID NO: 7) was isolated using the Arabidopsis API cDNA (SEQ ID NO: 1) as a probe.

In general terms, the invention relates to a non-naturally occurring seed plant that contains a first ectopically expressible nucleic acid molecule encoding a 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, and that is characterized by early reproductive development. As used herein, the term "characterized by early reproductive development," when used in reference to a non-naturally occurring seed plant of the invention, o* 25 means a non-naturally occurring seed plant that forms reproductive structures earlier than the time when reproductive structures form on a corresponding naturally occurring seed plant that is grown under the same conditions and that does not ecLopically express a floral WO 97/46078 PCTIUS96/09453 22 meristem identity gene product. For example, the reproductive structure of an angiosperm is a flower, and the reproductive structure of a coniferous plant is a cone. For a particular naturally occurring seed plant, reproductive development occurs at a well-defined time that depends, in part, on genetic factors as well as on environmental conditions, such as day length and temperature. Thus, given a defined set of environmental condition and lacking ectopic expression of a floral meristem identity gene product, a naturally occurring seed plant will undergo reproductive development at a relatively fixed time.

It is recognized that various transgenic plants that are characterized by early reproductive development have been described previously. Such transgenic plants, as discussed herein, are distinguishable from a non-naturally occurring seed plant of the invention or are explicitly excluded from the present invention. The product of a "late-flowering gene" can promote early reproductive development. However, a late flowering gene product is not a floral meristem identity gene product since it does not specify the conversion of shoot meristem to floral meristem in an angiosperm. Therefore, a transgenic plant expressing a late-flowering gene product is distinguishable from a non-naturally occurring seed plant of the invention. For example, a transgenic plant expressing the late-flowering gene, CONSTANS (CO), flowers earlier than the corresponding wild type plant, but does not contain an ectopically expressible nucleic acid molecule encoding a floral meristem identity gene WO 97/46078 PCTIUS96/09453 23 product (Putterill et al., Cell 80:847-857 (1995)).

Thus, the early-flowering transgenic plant described by Putterill et al. is not a non-naturally occurring seed plant as defined herein.

Early reproductive development also has been observed in a transgenic tobacco plant expressing an exogenous rice MADS domain gene. Although the product of the rice MADS domain gene promotes early reproductive development, it does not specify the identity of floral meristem and, thus, cannot convert shoot meristem to floral meristem in an angiosperm (Chung et al., Plant Mol. Biol. 26:657-665 (1994)). Therefore, an early-flowering transgenic plant containing this rice MADS domain gene, like an early-flowering transgenic plant containing CONSTANS, is distinguishable from an early-flowering non-naturally occurring seed plant of the invention.

Mutations in a class of genes known as "early-flowering genes" also produce plants characterized by early reproductive development. Such early-flowering genes include, for example, EARLY FLOWERING 1-3 (ELF1, ELF2, ELF3); EMBRYONIC FLOWER 1,2 (EMFI, EMF2); LONG HYPOCOTYL 1,2 (HY1, HY2); PHYTOCHROME B (PHYB), SPINDLY (SPY) and TERMINAL FLOWER (TFL) (Weigel, supra, 1995).

The wild type product of an early-flowering gene retards reproductive development and is distinguishable from a floral meristem identity gene product in that an early-flowering gene product does not promote conversion of shoot meristem to floral meristem in an angiosperm. A WO 97/46078 PCT/US96/09453 24 plant that flowers early due to the loss of an early-flowering gene product function is distinct from a non-naturally occurring seed plant of the invention characterized by early reproductive development since such a plant does not contain an ectopically expressible nucleic acid molecule encoding a floral meristem identity gene product.

An Arabidopsis plant having a mutation in the TERMINAL FLOWER (TFL) gene is characterized by early reproductive development and by the conversion of shoots to flowers (Alvarez et al., Plant J. 2:103-116 (1992), which is incorporated herein by reference). 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 lacks functional TFL, converts shoot meristem to floral meristem prematurely. 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 plant with an ectopically expressed floral meristem identity gene product. However, such a tfl mutant, in which a mutation in an endogenous TERMINAL FLOWER gene results in conversion of shoot meristem to floral meristem, is excluded explicitly from the present invention.

As used herein, the term "transgenic" refers to a seed plant that contains in its genome an exogenous WO 97/46078 PCTIUS96/09453 nucleic acid molecule, which can be derived from the same or a different plant species. The exogenous nucleic acid molecule can be a gene regulatory element such as a promoter, enhancer or other regulatory element or can contain a coding sequence, which can be linked to a heterologous gene regulatory element.

As used herein, the term "seed plant" means an angiosperm or a gymnosperm. The term "angiosperm," as used herein, means a seed-bearing plant whose seeds are borne in a mature ovary (fruit). An angiosperm commonly is recognized as a flowering plant. The term "gymnosperm," as used herein, means a seed-bearing plant with seeds not enclosed in an ovary.

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 single cotyledon, and a dicotyledonous angiosperm is an angiosperm having two cotyledons. 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 encompass a variety of flowering plants, including, for example, cereal plants, leguminous plants, oilseed plants, hardwood trees, fruit-bearing WO 97/46078 PCT/US96/09453 26 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 known as 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 also include alfalfa, birdsfoot trefoil, clover and sainfoin. An oilseed plant also is an angiosperm with seeds that are useful as a source of oil. Examples of oilseed plants include soybean, sunflower, rapeseed and cottonseed.

An angiosperm also can be a hardwood tree, which is a perennial woody plant that generally has a single stem (trunk). Examples of such trees include alder, ash, aspen, basswood (linden), beech, birch, cherry, cottonwood, elm, eucalyptus, hickory, locust, maple, oak, persimmon, poplar, sycamore, walnut and willow. Trees are useful, for example, as a source of pulp, paper, structural material and fuel.

An angiosperm also can be a fruit-bearing plant, which produces a mature, ripened ovary (usually containing seeds) that is suitable for human or animal consumption. For example, hops are a member of the mulberry family prized for their flavoring in malt WO 97/46078 PCTIUS96/09453 27 liquor. Fruit-bearing angiosperms also 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 commercially important ornamental flowers include rose, orchid, lily, tulip and chrysanthemum, snapdragon, camellia, carnation and petunia plants. The skilled artisan will recognize that the methods of the invention can be practiced using these or other angiosperms, as desired.

Gymnosperms encompass four divisions: cycads, ginkgo, conifers and gnetophytes. The conifers are the most widespread of living gymnosperms and frequently are cultivated for structural wood or for pulp or paper.

Conifers include redwood trees, pines, firs, spruces, hemlocks, Douglas firs, cypresses, junipers and yews.

The skilled artisan will recognize that the methods of the invention can be practiced with these and other gymnosperms.

As used herein, the term "non-naturally occurring seed plant" means a seed plant containing a genome that has been modified by man. A transgenic seed plant, for example, is a non-naturally occurring seed plant that contains an exogenous nucleic acid molecule and, therefore, has a genome that has been modified by man. Furthermore, a seed plant that contains, for example, a mutation in an endogenous floral meristem WO 97/46078

I'C

T

I )6/09453 28 identity gene regulatory element as a result of calculated exposure to a mutagenic agent also contains a genome that has been modified by man. In contrast, a seed plant containing a spontaneous or naturally occurring mutation is not a "non-naturally occurring seed plant" and, therefore, is not encompassed within the invention.

In general terms, the invention relates to a non-naturally occurring seed plant containing a first ectopically expressible nucleic adid molecule encoding a first 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. If desired, a non-naturally occurring seed plant of the invention can contain a second ectopically expressible nucleic acid molecule Sencoding a second floral meristem identity gene product Sthat is different from the first floral meristem identity o gene product, provided that the first or second nucleic o 20 acid molecule is not ectopically expressed due to a mutation in an endogenous TERMINAL FLOWER gene.

An ectopically expressible nucleic acid molecule encoding a floral meristem identity gene product can be expressed, as desired, either constitutively or 25 inducibly. Such an ectopically expressible nucleic acid molecule encoding a floral meristem identity gene product can be an endogenous floral meristem identity gene that has, for example, a mutation in a gene 1 egul.atory element. An ectopically express i.bl n l cl ic acid WO 97146078 PCT/US96/09453 29 molecule encoding a floral meristem identity gene product also can be an endogenous nucleic acid molecule encoding a floral meristem identity gene product that is linked to an exogenous, heterologous gene regulatory element that confers ectopic expression. In addition, an ectopically expressible nucleic acid molecule encoding a floral meristem identity gene product can be an exogenous nucleic acid molecule that encodes a floral meristem identity gene product under control of a heterologous gene regulatory element.

A non-naturally occurring seed plant of the invention can contain an endogenous floral meristem identity gene having a modified gene regulatory element.

The term "modified gene regulatory element," as used herein in reference to the regulatory element of a floral meristem identity gene, means a regulatory element having a mutation that results in ectopic expression of the linked endogenous floral meristem identity gene. 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. A modified gene regulatory element can have, for example, a nucleotide insertion, deletion or substitution that is produced, for example, by chemical mutagenesis using a mutagen such as ethylmethane sulfonate or by insertional mutagenesis using a transposable element. A modified gene regulatory element can be a functionally inactivated binding site for TFL or a functionally inactivated binding site for a gene product regulated by TFL, such that modification of WO 97/46078 [CTI/ 6/09453 the gene regulatory element results in ectopic expression of the linked floral meristem identity gene product, for example, in the shoot meristem of an angiosperm.

In general terms, the present invention also relates to a transgenic seed plant 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.

In general terms, the present invention also relates to a transgenic seed plant 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 S ectopically expressible nucleic acid molecule encoding a second floral meristem identity gene product, provided that the first nucleic acid molecule is not ectopically 20 expressed due to a mutation in an endogenous

TERMINAL

FLOWER gene.

In general terms, the present invention also relates to a S. transgenic seed plant containing a first exogenous ectopically expressible nucleic acid molecule encoding a first floral meristem identity gene product, provided that the first second nucleic acid molecule is not ectopically expressed due to a mutation in an endogenous TERMINAL FLOWER gene, and further containing a second WO 97/46078 PCT/US96/09453 31 exogenous ectopically expressible nucleic acid molecule encoding a second floral meristem identity gene product, where the first floral meristem identity gene product is different from the second floral meristem identity gene product.

As disclosed herein, ectopic expression of two different floral meristem identity gene products can be particularly useful. A transgenic Arabidopsis line constitutively expressing API under control of the cauliflower mosaic virus 35S promoter (see Example I) was crossed with a transgenic Arabidopsis line constitutively expressing LFY under control of the cauliflower mosaic virus 35S promoter (see Example III), and the resulting progeny were analyzed. A fraction of the progeny flowered were characterized by enhanced early reproductive development as compared to the early reproductive development of 35S-API transgenic lines or transgenic lines. PCR-based analyses demonstrated that all of the transgenic plants that were characterized by enhanced early reproductive development contained both the 35S-API and 35S-LFY transgenes. These results indicate that ectopic expression of the combination of AP1 and LFY in a seed plant can result in enhanced early reproductive development as compared to the early reproductive development obtained by ectopic expression of AP1 or LFY alone. Thus, by using a combination of two different floral meristem identity gene products, plant breeding, for example, can be accelerated further as compared to the use of a single floral meristem identity gene product.

WO 97/46078 PCTA 6/09453 32 A useful combination of first and second floral meristem identity gene products can be, for example, API and LFY, CAL and LFY, or API and CAL. A particularly useful combination of first and second floral meristem identity gene products is the combination of API with LFY, as disclosed above, or the combination of CAL with LFY. Where a transgenic seed plant of the invention contains first and second exogenous nucleic acid molecules encoding different floral meristem identity gene products, it will be recognized that the order of introducing the first and second nucleic acid molecules into the seed plant is not important for purposes of the present invention. Thus, a transgenic seed plant of the invention having, for example, AP1 as a first floral meristem identity gene product and LFY as a second floral meristem identity gene product is equivalent to a transgenic seed plant having LFY as a first floral

S

meristem identity gene product and API as a second floral meristem identity gene product.

20 In general, the invention also relates to methods of converting shoot meristem to floral meristem in an S. angiosperm by ectopically expressing a first ectopically expressible nucleic acid molecule encoding a first floral meristem identity gene product in the angiosperm. Thus, 25 the invention provides, for example, a method of converting shoot meristem to floral meristem in an S. 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 WO 9, j078 I'CTIUS96/09453 33 meristem identity gene product such as AP1, CAL or LFY, or a chimeric protein containing, in part, a floral meristem identity gene product, as disclosed below, is useful in converting shoot meristem to floral meristem.

As used herein, the term "introducing," when used in reference to a nucleic acid molecule and a seed plant such as an angiosperm or a gymnosperm, means transferring an exogenous nucleic acid molecule into the seed plant. For example, an exogenous nucleic acid molecule encoding a floral meristem identity gene product can be introduced into a seed plant by a variety of methods including Agrobacterium-mediated transformation or direct gene transfer methods such as electroporation or microprojectile-mediated transformation.

9 Transformation methods based upon the soil bacterium Agrobacterium tumefaciens, known as "agro-infection," are useful for introducing a nucleic acid molecule into a broad range of angiosperms and gymnosperms. The wild type form of Agrobacterium contains a Ti (tumor-inducing) plasmid that directs production of tumorigenic crown gall growth on host plants. Transfer of the tumor-inducing T-DNA region of the Ti plasmid to a plant genome requires the Ti o 25 plasmid-encoded virulence genes as well as T-DNA borders, which are a set of direct DNA repeats that delineate the region to be transferred. Agrobacterium-based vector is a modified form of a Ti plasmid, in which the tumor WO 97/46078 PCT/US96/09453 34 inducing functions are replaced by nucleic acid sequence of interest to be introduced into the plant host.

Current protocols for Agrobacterium-mediated transformation employ cointegrate vectors or, preferably, binary vector systems in which the components of the Ti plasmid are divided between a helper vector, which resides permanently in the Agrobacterium host and carries the virulence genes, and a shuttle vector, which contains the gene of interest bounded by T-DNA sequences. A variety of binary vectors are well known in the art and are commercially available from, for example, Clontech (Palo Alto, California). Methods of coculturing Agrobacterium with cultured plant cells or wounded tissue such as leaf tissue, root explants, hypocotyledons, stem pieces or tubers, for example, also are well known in the art (Glick and Thompson Methods in Plant Molecular Biology and Biotechnology, Boca Raton, FL: CRC Press (1993), which is incorporated herein by reference) Wounded cells within the plant tissue that have been infected by Agrobacterium can develop organs de novo when cultured under the appropriate conditions; the resulting transgenic shoots eventually give rise to transgenic plants containing the exogenous nucleic acid molecule of interest, as described in Example I.

Agrobacterium-mediated transformation has been used to produce a variety of transgenic seed plants (see, for example, Wang et al. (eds), Transformation of Plants and Soil Microorganisms, Cambridge, UK: University Press (1995), which is incorporated herein by reference). For WO 97/46078 PCT/US96/09453 example, Agrobacterium-mediated transformation can be used to produce transgenic crudiferous plants such as Arabidopsis, mustard, rapeseed and flax; transgenic leguminous plants such as alfalfa, pea, soybean, trefoil and white clover; and transgenic solanaceous plants such as eggplant, petunia, potato, tobacco and tomato. In addition, Agrobacterium-mediated transformation can be used to introduce exogenous nucleic acids into apple, aspen, belladonna, black currant, carrot, celery, cotton, cucumber, grape, horseradish, lettuce, morning glory, muskmelon, neem, poplar, strawberry, sugar beet, sunflower, walnut and asparagus plants (see, for example, Glick and Thompson, supra, 1993).

Microprojectile-mediated transformation also is a well known method of introducing an exogenous nucleic acid molecule into a variety of seed plant species. This method, first described by Klein et al., Nature 327:70-73 (1987), which is incorporated herein by reference, relies on microprojectiles such as gold or tungsten that are coated with the desired nucleic acid molecule by precipitation with calcium chloride, spermidine or PEG.

The microprojectile particles are accelerated at high speed into seed plant tissue using a device such as the Biolistic T M PD-1000 (Biorad, Hercules, California) Microprojectile-mediated delivery or "particle bombardment" is especially useful to transform seed plants that are difficult to transform or regenerate using other methods. Microprojectile-mediated transformation has been used, for example, to generate a WO 97/46078 PCT/US96/09453 36 variety of transgenic seed plant species, including cotton, tobacco, corn, hybrid poplar and papaya (see, for example, Glick and Thompson, supra, 1993). The transformation of important cereal crops such as wheat, oat, barley, sorghum and rice also has been'achieved using microprojectile-mediated delivery (Duan et al., Nature Biotech. 14:494-498 (1996); Shimamoto, Curr. Opin.

Biotech. 5:158-162 (1994), each of which is incorporated herein by reference). A rapid transformation regeneration system for the production of transgenic plants, such as transgenic wheat, in two to three months also can be useful in producing a transgenic seed plant of the invention (European Patent No. EP 0 709 462 A2, Application number 95870117.9, filed 25 October 1995, which is incorporated herein by reference).

Thus, a variety of methods for introducing a nucleic acid molecule into a seed plant are well known in the art. Important crop species such as rice, for example, have been transformed using microprojectile delivery, Agrobacterium-mediated transformation or protoplast transformation (Hiei et al., The Plant J.

6(2):271-282 (1994); Shimamoto, Science 270:1772-1773 (1995), each of which is incorporated herein by reference). Fertile transgenic maize has been obtained, for example, by microparticle bombardment (see Wang et al., supra, 1995). As discussed above, barley, wheat, oat and other small-grain cereal crops also have been transformed, for example, using microparticle bombardment (see Wang et al., supra, 1995).

WO 97/46078 PCT/US96/09453 37 Methods of transforming forest trees including both angiosperms and gymnosperms also are well known in the art. Transgenic angiosperms such as members of the genus Populus, which includes aspens and poplars, have been generated using Agrobacterium-mediated transformation, for example. In addition, transgenic Populus and sweetgum, which are of interest for biomass production for fuel, also have been produced. Transgenic gymnosperms, including conifers such as white spruce and larch, also have been obtained, for example, using microprojectile bombardment (Wang et al., supra, 1995).

The skilled artisan will recognize that Agrobacterium-mediated or microprojectile-mediated transformation, as disclosed herein, or other methods known in the art can be used to introduce a nucleic acid molecule encoding a floral meristem identity gene product into a seed plant according to the methods of the invention.

The term "converting shoot meristem to floral meristem," as used herein, means promoting the formation of flower progenitor tissue where shoot progenitor tissue otherwise would be formed in the angiosperm. 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 (see, for example, Mandel and Yanofsky, Plant Cell 7:1763-1771 (1995), which is incorporated herein by reference or Weigel and Nilsson, supra, 1995).

WO 97/46078 PCT/US96/09453 38 Provided herein are 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, where the first floral meristem identity gene product is different from the second floral meristem identity gene product. As discussed above, first and second floral meristem identity gene products useful in converting shoot meristem to floral meristem in an angiosperm can be, for example, AP1 and LFY, CAL and LFY, or AP1 and CAL.

Also provided herein are methods of promoting early reproductive development in a seed plant by ectopically expressing a first nucleic acid molecule encoding a first floral meristem identity gene product in the seed plant, provided that the first nucleic acid molecule is not ectopically expressed due to a mutation in an endogenous TERMINAL FLOWER gene. For example, the invention provides a method of promoting early reproductive development in a seed plant by introducing an ectopically expressible nucleic acid molecule encoding a floral meristem identity gene product into the seed plant, thus producing a transgenic seed plant. 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, as disclosed below, is useful in methods of promoting early reproductive development.

WO 97/46078 PCT/US96/09453 39 The term "promoting early reproductive development," as used herein in reference to a seed plant, means promoting the formation of a reproductive structure earlier than the time when a reproductive structure would form on a corresponding seed plant that is grown under the same conditions and that does not ectopically express a floral meristem identity gene product. As discussed above, the time when reproductive structures form on a particular seed plant that does not ectopically express a floral meristem identity gene product is relatively fixed and depends, in part, on genetic factors as well as environmental conditions, such as day length and temperature. Thus, given a defined set of environmental conditions, a naturally occurring angiosperm, for example, will flower at a relatively fixed time. Similarly, given a defined set of environmental conditions, a naturally occurring coniferous gymnosperm, for example, will produce cones at a relatively fixed time.

As disclosed herein, ectopic expression of a nucleic acid molecule encoding a floral meristem identity gene product in an angiosperm converts shoot meristem to floral meristem in the angiosperm. Furthermore, ectopic expression of a nucleic acid molecule encoding a floral meristem identity gene product such as API, CAL or LFY in an angiosperm prior to the time when endogenous floral meristem identity gene products are expressed in the angiosperm can convert shoot meristem to floral meristem precociously, resulting in early reproductive development in the angiosperm, as indicated by early flowering. In WO 97/46078 PCT/US96/09453 the same manner, ectopic expression of a nucleic acid molecule encoding AP1, CAL, or LFY, for example, in a gymnosperm prior to the time when endogenous floral meristem identity gene products are expressed in the gymnosperm results in early reproductive development in the gymnosperm.

For a given seed plant species and particular set of growth conditions, constitutive expression of a floral meristem identity gene product results in a relatively invariant time of early reproductive development, which is the earliest time when all factors necessary for reproductive development are active. For example, as shown in Example ID, constitutive expression of API in transgenic Arabidopsis plants grown under "long-day" light conditions results in early reproductive development at day 10 as compared to the normal time of reproductive development, which is day 18 in non-transgenic Arabidopsis plants grown under the same conditions. Thus, under these conditions, day 10 is the relatively invariant time of early reproductive development for Arabidopsis transgenics that constitutively express a floral meristem identity gene product.

However, in addition to methods of constitutively expressing a floral meristem identity gene product, the present invention provides methods of selecting the time of early reproductive development. As disclosed herein, floral meristem gene product expression or activity can be regulated in response to an inducing WO 97/46078 PCT/US96/09453 41 agent or cognate ligand, for example, such that the time of early reproductive development can be selected. For example, in Arabidopsis transgenics grown under the conditions described above, the time of early reproductive development need not necessarily be the relatively invariant day 10 at which early reproductive development occurs as a consequence of constitutive floral meristem identity gene product expression. If floral meristem identity gene product expression is rendered dependent upon the presence of an inducing agent, early reproductive development can be selected to occur, for example, on day 14, by contacting the seed plant with an inducing agent on or slightly before day 14.

Thus, the present invention provides recombinant nucleic acid molecules, transgenic seed plant containing such recombinant nucleic acid molecules and methods for selecting the time of early reproductive development. These methods allow a farmer or horticulturist, for example, to determine the time of early reproductive development. The methods of the invention can be useful, for example, in allowing a grower to respond to an approaching storm or impending snap-freeze by selecting the time of early reproductive development such that the crop can be harvested before being harmed by the adverse weather conditions. The methods of the invention for selecting the time of early reproductive development also can be useful to spread out the time period over which transgenic seed plants are ready to be harvested. For example, the methods of the 42 invention can be used to increase floral meristem identity gene product expression in different crop fields at different times, resulting in a staggered time of harvest for the different fields.

Thus, the present invention provides a recombinant nucleic acid molecule containing an inducible regulatory element operably linked to a nucleic acid molecule encoding a floral meristem identity gene product.

As disclosed herein, a recombinant nucleic acid molecule of the invention can contain an inducible regulatory element such as a copper inducible element, tetracycline inducible element, ecdysone inducible element or heat shock inducible element.

e 9*. e a ooo0 «~o *~o 43 The term "recombinant" nucleic acid molecule," as used herein, means a non-naturally occurring nucleic acid molecule that has been manipulated in vitro such that it is genetically distinguishable from a naturally occurring nucleic acid molecule. A recombinant nucleic acid molecule of the invention comprises two nucleic acid molecules that have been manipulated in vitro such that the two nucleic acid molecules are operably linked.

As used herein, the term "inducible regulatory element" means a nucleic acid molecule that confers conditional expression upon an operably linked nucleic acid molecule, where expression of the operably linked nucleic acid molecule is increased in the presence of a particular inducing agent as compared to expression of invention can be used to increase floral meristem identity gene product expression in different crop fields at different times, resulting in a staggered time of harvest for the different fields.

too* 4 oa S6 *I 4

S

oooo ft***o WO 97/46078 PCT/US96/09453 44 the nucleic acid molecule in the absence of the inducing agent. In a method of the invention, a useful inducible regulatory element has the following characteristics: confers low level expression upon an operably linked nucleic acid molecule in the absence of an inducing agent; confers high level expression upon an operably linked nucleic acid molecule in the presence of an appropriate inducing agent; and utilizes an inducing agent that does not interfere substantially with the normal physiology of a transgenic seed plant treated with the inducing agent. It is recognized, for example, that, subsequent to introduction into a seed plant, a particularly useful inducible regulatory element is one that confers an extremely low level of expression upon an operably linked nucleic acid molecule in the absence of inducing agent. Such an inducible regulatory element is considered to be tightly regulated.

The term "operably linked," as used in reference to a regulatory element, such as a promoter or inducible regulatory element, and a nucleic acid molecule encoding a floral meristem identity gene product, means that the regulatory element confers regulated expression upon the operably linked nucleic acid molecule encoding the floral meristem identity gene product. Thus, the term operably linked, as used herein in reference to an inducible regulatory element and a nucleic acid molecule encoding a floral meristem identity gene product, means that the inducible regulatory element is linked to the nucleic acid molecule encoding a floral meristem identity gene product such that the inducible regulatory element WO 97/46078 PCT/US96/09453 increases expression of the floral meristem identity gene product in the presence of the appropriate inducing agent. It is recognized that two nucleic acid molecules that are operably linked contain, at a minimum, all elements essential for transcription, including, for example, a TATA box. One skilled in the art knows, for example, that an inducible regulatory element that lacks minimal promoter elements can be combined with a nucleic acid molecule having minimal promoter elements and a nucleic acid molecule encoding a floral meristem identity gene product such that expression of the floral meristem identity gene product can be increased in the presence of the appropriate inducing agent.

A particularly useful inducible regulatory element can be, for example, a copper-inducible promoter (Mett et al., Proc. Natl. Acad. Sci. USA 90:4567-4571 (1993), which is incorporated herein by reference); tetracycline-inducible regulatory element (Gatz et al., Plant J. 2:397-404 (1992); R6der et al., Mol. Gen. Genet.

243:32-38 (1994), each of which is incorporated herein by reference); ecdysone inducible element (Christopherson et al., Proc. Natl. Acad. Sci. USA 89:6314-6318 (1992), which is incorporated herein by reference); or heat shock inducible element (Takahashi et al., Plant Physiol.

99:383-390 (1992), which is incorporated herein by reference). Another useful inducible regulatory element can be a lac operon element, which is used in combination with a constitutively expressed lac repressor to confer, for example, IPTG-inducible expression, as described by WO 97/46078 PCT/US96/09453 46 Wilde et al., (EMBO J. 11:1251-1259 (1992), which is incorporated herein by reference).

An inducible regulatory element useful in a method of the invention also can be, for example, a nitrate-inducible promoter derived from the spinach nitrite reductase gene (Back et al., Plant Mol.

Biol. 17:9 (1991), which is incorporated herein by reference) or a light-inducible promoter, such as that associated with the small subunit of RuBP carboxylase or the LHCP gene families (Feinbaum et al., Mol. Gen. Genet.

226:449 (1991); Lam and Chua, Science 248:471 (1990), each of which is incorporated herein by reference). An inducible regulatory element useful in constructing a transgenic seed plant also can be a salicylic acid inducible element (Uknes et al., Plant Cell 5:159-169 (1993); Bi et al., Plant J. 8:235-245 (1995), each of which is incorporated herein by reference) or a plant hormone-inducible element (Yamaguchi-Shinozaki et al., Plant Mol. Biol. 15:905 (1990); Kares et al., Plant Mol.

Biol. 15:225 (1990), each of which is incorporated herein by reference). A human glucocorticoid response element also is an inducible regulatory element that can confer hormone-dependent gene expression in seed plants (Schena et al., Proc. Natl. Acad. Sci. USA 88:10421 (1991), which is incorporated herein by reference).

An inducible regulatory element that is particularly useful for increasing expression of a floral meristem identity gene product in a transgenic seed plant of the invention is a copper inducible regulatory element WO 97/46078 PCT/US96/09453 47 (see, for example, Mett et al., supra, 1993). Thus, the invention provides a recombinant nucleic acid molecule comprising a copper inducible regulatory element operably linked to a nucleic acid molecule encoding a floral meristem identity gene product and a transgenic seed plant containing such a recombinant nucleic acid molecule. Copper, which is a natural part of the nutrient environment of a seed plant, can be used to increase expression of a nucleic acid molecule encoding a floral meristem identity gene product operably linked to a copper inducible regulatory element. For example, an ACE1 binding site in conjunction with constitutively expressed yeast ACE1 protein confers copper inducible expression upon an operably linked nucleic acid molecule.

The ACE1 protein, a metalloresponsive transcription factor, is activated by copper or silver ions, resulting in increased expression of a nucleic acid molecule operably linked to an ACE1 element.

Such a copper inducible regulatory element can be an ACE1 binding site from the metallothionein gene promoter (SEQ ID NO: 21; Furst et al., Cell 55:705-717 (1988), which is incorporated herein by reference). For example, the ACE1 binding site can be combined with the base-pair domain A of the cauliflower mosaic virus promoter and operably linked to a nucleic acid molecule encoding AP1, CAL or LFY to produce a recombinant nucleic acid molecule of the invention. In a transgenic seed plant constitutively expressing ACE1 under control of such a modified CaMV 35S promoter, for example, copper inducible expression is conferred upon an operably linked WO 97/46078 PCT/US96/09453 48 nucleic acid molecule encoding a floral meristem identity gene product.

The expression of a nucleic acid encoding a floral meristem identity gene product operably linked to a copper inducible regulatory element, such as 5'-AGCTTAGCGATGCGTCTTTTCCGCTGAACCGTTCCAGCAAAAAAGACTAG-3' (SEQ ID NO: 21), can be increased in a transgenic seed plant grown under copper ion-depleted conditions, for example, and contacted with 50 pM copper sulfate in a nutrient solution or with 0.5 AM copper sulfate applied by foliar spraying of the transgenic seed plant (see, for example, Mett et al., supra, 1993). A single application of 0.5 AM copper sulfate can be sufficient to sustain increased floral meristem identity gene product expression over a period of several days. If desired, a transgenic seed plant of the invention also can be contacted with multiple applications of an inducing agent such as copper sulfate.

An inducible regulatory element also can confer tetracycline-dependent floral meristem identity gene expression in a transgenic seed plant of the invention.

Thus, the present invention provides a recombinant nucleic acid molecule comprising a tetracycline inducible regulatory element operably linked to a nucleic acid molecule encoding a floral meristem identity gene product as well as a transgenic seed plant into which such a recombinant nucleic acid molecule has been introduced.

A tetracycline inducible regulatory element is particularly useful for conferring tightly regulated gene WO 97/46078 PCT/US96/09453 49 expression as indicated by the observation that a phenotype that results from even low amounts of a gene product expression is suppressed from such an inducible system in the absence of inducing agent (see, for example, R6der et al., supra, 1994).

A transgenic seed plant constitutively expressing Tnl0-encoded Tet repressor (TetR), for example, can be contacted with tetracycline to increase expression of a nucleic acid molecule encoding a floral meristem identity gene product operably linked to the cauliflower mosaic virus promoter containing several tet operator sequences (5'-ACTCTATCAGTGATAGAGT-3'; SEQ ID NO: 22) positioned close to the TATA box (see, for example, Gatz, Meth. Cell Biol. 50:411-424 (1995), which is incorporated herein by reference; Gatz et al., supra, 1992). Such a tetracycline-inducible system can increase expression of an operably linked nucleic acid molecule as much as 200 to 500-fold in a transgenic angiosperm or gymnosperm of the invention.

A high level of Tet repressor expression (about 1 x 106 molecules per cell) is critical for tight regulation. Thus, a seed plant preferably is transformed first with a plasmid encoding the Tet repressor, and screened for high level expression. For example, plasmid pBinTet (Gatz, supra, 1995) contains the Tet repressor coding region, which is expressed under control of the CaMV 35S promoter, and the neomycin phosphotransferase gene for selection of transformants. To screen transformants for a high level of Tet repressor WO 97/46078 PCT/US96/09453 expression, a plasmid containing a reporter gene under control of a promoter with tet operators, such as pTX-Gus-int (Gatz, supra, 1995), can be transiently introduced into a seed plant cell and assayed for activity in the presence and absence of tetracycline.

High P-glucouronidase (GUS) expression that is dependent on the presence of tetracycline is indicative of high Tet repressor expression.

A particularly useful tetracycline inducible regulatory element is present in plasmid pBIN-HygTX, which has a CaMV 35S promoter, into which three tet operator sites have been inserted, and an octopine synthase polyadenylation site (Gatz, supra, 1995). A multiple cloning site between the promoter and polyadenylation signal in pBIN-HygTX allows for convenient insertion of a nucleic acid molecule encoding the desired floral meristem identity gene product, and the hygromycin phosphotransferase gene allows for selection of transformants containing the construct. In a preferred embodiment of the invention, previously selected Tet repressor positive cells are transformed with a plasmid such as pBIN-HygTX, into which a nucleic acid molecule encoding a floral meristem identity gene product has been inserted.

To increase floral meristem identity gene product expression using a tetracycline-inducible regulatory element, a transgenic seed plant of the invention can be contacted with tetracycline or, preferably, with chlor-tetracycline (SIGMA), which is a WO 97/46078 PCT/US96/09453 51 more efficient inducer than tetracycline. In addition, a useful inducing agent can be a tetracycline analog that binds the Tet repressor to function as an inducer but that does not act as an antibiotic (Gatz, supra, 1995).

A transgenic seed plant of the invention can be contacted, for example, by watering with about 1 mg/liter chlor-tetracycline or tetracycline. Similarly, a plant grown in hydroponic culture can be contacted with a solution containing about 1 mg/liter chlor-tetracycline or tetracycline (Gatz, supra, 1995). If desired, a transgenic angiosperm or gymnosperm can be contacted repeatedly with chlor-tetracycline or tetracycline every other day for about 10 days (RBder et al., supra, 1994).

Floral meristem identity gene product expression is increased efficiently at a tetracycline concentration that does not inhibit the growth of bacteria, indicating that the use of tetracycline as an inducing agent will not present environmental concerns.

An ecdysone inducible regulatory element also can be useful in practicing the methods of the invention.

For example, an ecdysone inducible regulatory element can contain four copies of an ecdysone response element having the sequence 5'-GATCCGACAAGGGTTCAATGCACTTGTCA-3' (EcRE; SEQ ID NO: 23) as described in Christopherson et al., supra, 1992. In a transgenic seed plant into which a nucleic acid encoding an ecdysone receptor has been introduced, an ecdysone inducible regulatory element can confer ecdysone-dependent expression on a nucleic acid molecule encoding a floral meristem identity gene product. An appropriate inducing agent for increasing WO 97/46078 PCT/US96/09453 52 expression of a nucleic acid molecule operably linked to an ecdysone inducible regulatory element can be, for example, a-ecdysone, 20-hydroxyecdysone, polypodine B, ponasterone A, muristerone A or RH-5992, which is an ecdysone agonist that mimics 20-hydroxyecdysone (see, for example, Kreutzweiser et al., Ecotoxicol. Environ. Safety 28:14-24 (1994), which is incorporated herein by reference and Christopherson et al., supra, 1992).

Methods for determining an appropriate inducing agent for use with an ecdysone inducible regulatory element are well known in the art. As disclosed herein, compound RH-5992 can be a particularly useful inducing agent for increasing floral meristem gene product expression in a transgenic seed plant containing an ecdysone inducible regulatory element.

An inducible regulatory element also can be derived from the promoter of a heat shock gene, such as HSP81-1 (SEQ ID NO: 24; Takahashi, supra, 1992). Thus, the invention also provides a recombinant nucleic acid molecule comprising a heat shock inducible regulatory element operably linked to a nucleic acid molecule encoding a floral meristem identity gene product and a transgenic seed plant containing such a recombinant nucleic acid molecule. The HSP81-1 promoter (SEQ ID NO: 24) confers low level expression upon an operably linked nucleic acid molecule in parts of roots under unstressed conditions and confers high level expression in most Arabidopsis tissues following heat shock (see, for example, Yabe et al., Plant Cell Physiol.

35:1207-1219 (1994), which is incorporated herein by WO 97/46078 PCT/US96/09453 53 reference). After growth of Arabidopsis at 23 0 C, a single heat shock treatment at 37 0 C for two hours is sufficient to induce expression of a nucleic acid molecule operably linked to the HSP81-1 gene regulatory element (see Ueda et al., Mol. Gen. Genet. 250:533-539 (1996), which is incorporated herein by reference).

The use of a heat shock inducible regulatory element is particularly useful for a transgenic seed plant of the invention grown in an enclosed environment such as a green house, where temperature can be readily manipulated. The use of a heat shock inducible regulatory element especially is applicable to a transplantable or potted transgenic seed plant of the invention, which can be moved conveniently from an environment having a low temperature to an environment having a high temperature. A transgenic angiosperm or gymnosperm of the invention containing a recombinant nucleic acid molecule comprising a HSP81-1 heat shock regulatory element operably linked to a nucleic acid molecule encoding a floral meristem identity gene product also can be induced, for example, by altering the ambient temperature, watering with heated water or submersing the transgenic seed plant in a sealed plastic bag into a heated water bath (see, for example, Ueda et al., supra, 1996).

A recombinant nucleic acid molecule of the invention comprising an inducible gene regulatory element can be expressed variably in different lines of transgenic seed plants. In some transgenic lines, for WO 97/46078 PCT/ )6/09453 54 example, leaky expression of the introduced recombinant nucleic acid molecule can occur in the absence of the appropriate inducing agent due to phenomena such as position effects (see, for example, Ueda et al., supra, 1996). Thus, a transgenic seed plant containing a recombinant nucleic acid molecule comprising an inducible gene regulatory element operably linked to a nucleic acid encoding a floral meristem identity gene product can be screened, if desired, to obtain a particular transgenic seed plant in which expression of the operably linked nucleic acid molecule is desirably low in the absence of the appropriate inducing agent.

Also provided is a method of converting shoot meristem to floral meristem in an angiosperm by introducing into the angiosperm a recombinant nucleic acid molecule comprising an inducible eae$ regulatory element operably linked to a nucleic acid molecule according to the invention S...to produce a transgenic angiosperm, and contacting the transgenic angiosperm with an inducing agent, thereby increasing expression of the floral meristem identity gene product and converting shoot meristem to floral meristem in the transgenic angiosperm. In such a method of the invention, the inducible regulatory element can be, for example, a copper inducible element, tetracycline inducible element, ecdysone inducible element or heat shock inducible element.

55 Also provided is a method of promoting early reproductive development in a seed plant such as an angiosperm or gymnosperm by introducing into the seed plant a recombinant nucleic acid molecule comprising an inducible regulatory element operably linked to a nucleic acid molecule according to the invention to produce a transgenic seed plant, and contacting the transgenic seed plant with an inducing agent, thereby increasing expression of the floral meristem identity gene product and promoting early reproductive development in the transgenic seed plant. In a method of the invention for promoting early reproductive development in a seed plant, the inducible regulatory element can be, for example, a copper inducible element, tetracycline inducible element, ecdysone inducible element or heat shock inducible element.

The term "inducing agent," as used herein, means a substance or condition that effects increased expression of a nucleic acid molecule operably linked to a particular inducible regulatory element as compared to the level of expression of the nucleic acid molecule in the absence of the inducing agent. An inducing agent can be, for example, 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 increases expression of a nucleic acid molecule operably linked to a particular inducible eo* regulatory element. An example of such an inducing agent

S

ooooo WO 97/46078 PCT/US96/09453 56 is a compound such as copper sulfate, tetracycline or an ecdysone. An inducing agent also can be a condition such as heat of a certain temperature or light of a certain wavelength. When used in reference to a particular inducible regulatory element, an "appropriate" inducing agent means an inducing agent that results in increased expression of a nucleic acid molecule operably linked to the particular inducible regulatory element.

An inducing agent of the invention can be used alone or in solution or can be used in conjunction with an acceptable carrier that can serve to stabilize the inducing agent or to promote absorption of the inducing agent by a seed plant. If desired, a transgenic seed plant of the invention can be contacted with an inducing agent in combination with an unrelated substance such as a plant nutrient, pesticide or insecticide.

One skilled in the art can readily determine the optimum concentration of an inducing agent needed to produce increased expression of a nucleic acid molecule operably linked to an inducible regulatory element in a transgenic seed plant of the invention. For conveniently determining the optimum concentration of inducing agent from a range of useful concentrations, one skilled in the art can operably link the particular inducible regulatory element to a nucleic acid molecule encoding a reporter gene product such as -glucouronidase (GUS) and assay for reporter gene product activity in the presence of various concentrations of inducing agent (see, for example, WO 97/46078 PCT/US96/09453 57 Jefferson et al., EMBO J. 6:3901-3907 (1987), which is incorporated herein by reference) As used herein, the term ,contacting," in reference to a transgenic seed plant of the invention, means exposing the transgenic seed plant to an inducing agent, or to a cognate ligand as disclosed below, such that the agent can induce expression of a nucleic acid molecule operably linked to the particular inducible regulatory element. A transgenic seed plant such as an angiosperm or gymnosperm, which contains a recombinant nucleic acid molecule of the invention, can be contacted with an inducing agent in a variety of manners.

Expression of a floral meristem identity gene product can be increased conveniently, for example, by spraying a transgenic seed plant with an aqueous solution containing an appropriate inducing agent or by adding an appropriate inducing agent to the water supply of a transgenic seed plant grown using irrigation or to the water supply of a transgenic seed plant grown hydroponically. A transgenic seed plant containing a recombinant nucleic acid molecule of the invention also can be contacted by spraying the seed plant with an inducing agent in aerosol form. In addition, a transgenic seed plant can be contacted with an appropriate inducing agent by adding the agent to the soil or other solid nutrient media in which the seed plant is grown, whereby the inducing agent is absorbed into the seed plant. Other modes of contacting a transgenic seed plant with an inducing agent, such as injecting or immersing the seed plant in a solution containing an inducing agent, are well known in the art.

WO 97/46078 PCT/US96/09453 58 For an inducing agent that is temperature or light, for example, contacting can be effected by altering the temperature or light to which the transgenic seed plant is exposed, or, if desired, by moving the transgenic seed plant from an environment of one temperature or light source to an environment having the appropriate inducing temperature or light source.

If desired, a transgenic seed plant of the invention can be contacted individually with an inducing agent. Furthermore, a group of transgenic seed plants that, for example, are located together in a garden plot, hot house or field, can be contacted en masse with an inducing agent, such that floral meristem identity gene product expression is increased coordinately in all transgenic seed plants of the group.

A transgenic seed plant of the invention can be contacted with an inducing agent using one of several means. For example, a transgenic seed plant can be contacted with an inducing agent by non-automated means such as with a hand held spraying apparatus. Such manual means can be useful when the methods of the invention are applied to particularly delicate or valuable seed plant varieties or when it is desirable, for example, to promote early reproductive development in a particular transgenic seed plant without promoting early reproductive development in a neighboring transgenic seed plant. Furthermore, a transgenic seed plant of the invention can be contacted with an inducing agent by mechanical means such as with a conventional yard WO 97/46078 PCT/US96/09453 59 "sprinkler" for a transgenic seed plant grown, for example, in a garden; a mechanical spraying system in a green house; traditional farm machinery for spraying field crops; or "crop dusting" for conveniently contacting an entire field of transgenic seed plants with a particulate or gaseous inducing agent. The skilled practitioner, whether home gardener or commercial farmer, recognizes that these and other manual or mechanical means can be used to contact a transgenic seed plant with an inducing agent according to the methods of the invention.

Furthermore, it is recognized that a transgenic seed plant of the invention can be contacted with a single treatment of an inducing agent or, if desired, can be contacted with multiple applications of the inducing agent. In a preferred embodiment of the invention, a transgenic seed plant of the invention is contacted once with an inducing agent to effectively increase floral meristem identity gene product expression, thereby promoting early reproductive development in the transgenic seed plant. Similarly, a transgenic angiosperm of the invention preferably is contacted once with an inducing agent to effectively increase floral meristem identity gene product expression and convert shoot meristem to floral meristem in the transgenic angiosperm.

A single application of an inducing agent is preferable when a transient increase in floral meristem identity gene product expression from a recombinant WO 97/46078 PCT/US96/09453 nucleic acid molecule of the invention promotes irreversible early reproductive development in a seed plant. In many seed plant species, early reproductive development is irreversible. Transient expression of a floral meristem identity gene product from an introduced recombinant nucleic acid molecule, for example, results in sustained ectopic expression of endogenous floral meristem identity gene products, resulting in irreversible early reproductive development. For example, ectopic expression of AP1 in a transgenic plant induces endogenous LFY gene expression, and ectopic expression of LFY induces endogenous AP1 gene expression (Mandel and Yanofsky, Nature 377:522-524 (1995), which is incorporated herein by reference; Weigel and Nilsson, supra, 1995). Genetic studies also indicate that CAL can act directly or indirectly to increase expression of AP1 and LFY. Thus, ectopic expression of CAL from an exogenous nucleic acid molecule, for example, can induce endogenous API and LFY expression (see Bowman et al., supra, 1993). Enhanced expression of endogenous AP1, LFY or CAL following a transient increase in expression of an introduced floral meristem identity gene product induced by a single application of an inducing agent can make repeated applications of an inducing agent unnecessary.

In some seed plants, however, such as angiosperms characterized by the phenomenon of floral reversion, repeated applications of the inducing agent can be desirable. In species such as impatiens, an initiated flower can revert into a shoot such that the center of the developing flower behaves as an WO 91. 78 PCT/US96/09453 61 indeterminate shoot (see, for example, Battey and Lyndon, Ann. BQt. 61:9-16 (1988), which is incorporated by reference herein). Thus, to prevent floral reversion in species such as impatiens, repeated applications of an inducing agent can be useful. Repeated applications of an inducing agent, as well as single applications, are encompassed within the scope of the present invention.

In general terms, there is envisaged a nucleic acid molecule encoding a chimeric protein, which comprises a nucleic acid molecule encoding a floral meristem identity gene product such as AP1, CAL or LFY linked in frame to a nucleic acid molecule encoding a ligand binding domain.

Expression of a chimeric protein of the invention in a seed plant is useful because the ligand binding domain renders the activity of a linked gene product dependent on the presence of cognate ligand. Specifically, in a chimeric protein of the invention, floral meristem gene product activity is increased in the presence of cognate ligand, as compared to activity in the absence of cognate ligand.

A nucleic acid molecule encoding a chimeric protein of the invention comprises a nucleic acid molecule encoding a floral meristem identity gene product, such as a nucleic acid molecule having the nucleic acid sequence SEQ ID NO: 1, SEQ ID NO: 9 or SEQ ID NO: 15, which encodes AP1, CAL or LFY, respectively, any of which is linked in frame to a nucleic acid molecule encoding a ligand binding domain. The expression of such a nucleic acid molecule results in the 62 production of a chimeric protein containing a floral meristem identity gene product fused to a ligand binding domain.

Also envisaged in general terms is transgenic seed plant, such as angiosperm or gymnosperm, that contains a nucleic acid molecule encoding a chimeric protein of the invention, for example, a transgenic seed plant containing a nucleic acid molecule encoding a chimeric protein, which comprises a nucleic acid molecule encoding AP1, CAL or WY linked in frame to a nucleic acid molecule encoding a ligand binding domain. A particularly useful transgenic seed plant contains a nucleic acid molecule encoding AP1 linked in frame to a nucleic acid molecule encoding an ecdysone receptor ligand binding domain or a glucocorticoid receptor ligand binding domain.

This could also be a transgenic seed plant containing a nucleic acid molecule encoding a chimeric protein, which comprises a nucleic acid molecule encoding CAL linked in frame to a nucleic acid molecule encoding an ecdysone receptor ligand binding domain or a glucocorticoid receptor ligand binding domain. In addition, there is provided a transgenic seed plant containing a nucleic acid molecule encoding a chimeric protein, which comprises a nucleic acid molecule encoding LFY linked in frame to a nucleic acid molecule encoding WO 97/46078 PCT/US96/09453 63 an ecdysone receptor ligand binding domain or a glucocorticoid receptor ligand binding domain.

Any floral meristem identity gene product, as defined herein, is useful in a chimeric protein of the invention. Thus, a nucleic acid molecule encoding Arabidopsis thaliana API (SEQ ID NO: Brassica oleracea AP1 (SEQ ID NO: Brassica oleracea var.

Botrytis AP1 (SEQ ID NO: 8) or Zea mays AP1 (SEQ ID NO: 10), each of which have activity in converting shoot meristem to floral meristem, can be used to construct a nucleic acid molecule encoding a chimeric protein of the invention. Similarly, a nucleic acid molecule encoding, for example, Arabidopsis thaliana CAL (SEQ ID NO: Brassica oleracea CAL (SEQ ID NO: 12), or a nucleic acid molecule encoding Arabidopsis thaliana LFY (SEQ ID NO: 16) is useful when linked in frame to a nucleic acid molecule encoding a ligand binding domain to produce a nucleic acid molecule encoding a ligand-dependent chimeric protein of the invention.

A ligand binding domain useful in a chimeric protein of the invention is a domain that, when fused in frame to a heterologous gene product, renders the activity of the fused gene product dependent on cognate ligand such that the activity of the fused gene product is increased in the presence of cognate ligand as compared to its activity in the absence of ligand. Such a ligand binding domain can be a steroid binding domain such as the ligand binding domain of an ecdysone receptor, glucocorticoid receptor, estrogen receptor, WO 97/46078 PCT/US96/09453 64 progesterone receptor, androgen receptor, thyroid receptor, vitamin D receptor or retinoic acid receptor.

A particularly useful ligand binding domain is the ecdysone receptor ligand binding domain contained within amino acids 329 to 878 of the Drosophila ecdysone receptor (SEQ ID NO: 18); Koelle et al., Cell 67:59-77 (1991); Thummel, Cll 83:871-877 (1995), each of which is incorporated herein by reference) or a glucocorticoid receptor ligand binding domain, encompassed, for example, within amino acids 512 to 795 of the rat glucocorticoid receptor (SEQ ID NO: 20; Miesfeld et al., Cell 46:389-399 (1986), which is incorporated herein by reference) A chimeric protein of the invention containing an ecdysone receptor ligand binding domain has floral meristem identity gene product activity that can be increased in the presence of ecdysone ligand. Similarly, a chimeric protein of the invention containing a glucocorticoid receptor ligand binding domain has floral meristem identity gene product activity that is increased in the presence of glucocorticoid ligand. It is well known that in a chimeric protein containing a heterologous gene product such as adenovirus E1A, c-myc, c-fos, the HIV-1 Rev transactivator, MyoD or maize regulatory factor R fused to the rat glucocorticoid receptor ligand binding domain, activity of the fused heterologous gene product can be increased by glucocorticoid ligand (Eilers et al., Nature 340:66 (1989); Superti-Furga et al., Proc. Natl. Acad. Sci..

U.S.A. 88:5114 (1991); Hope et al., Proc. Natl. Acad.

Sci.. U.S.A. 87:7787 (1990); Hollenberg et al., Proc.

WO 97/46078 PCT/US96/09453 Natl. Acad. Sci.. U.S.A. 90:8028 (1993), each of which is incorporated herein by reference).

A nucleic acid molecule encoding a chimeric protein of the invention can be introduced into a seed plant where, under appropriate conditions, the chimeric protein is expressed. In such a transgenic seed plant, floral meristem identity gene product activity can be increased by contacting the transgenic seed plant with cognate ligand. For example, activity of a heterologous protein fused to a rat glucocorticoid receptor ligand binding domain (amino acids 512 to 795) expressed under the control of the constitutive cauliflower mosaic virus promoter in Arabidopsis was low in the absence of glucocorticoid ligand; whereas, upon contacting the transformed plants with a synthetic glucocorticoid, dexamethasone, activity of the protein was increased greatly (Lloyd et al., Science 266:436-439 (1994), which is incorporated herein by reference). As disclosed herein, a ligand binding domain fused to a floral meristem identity gene product renders the activity of a fused floral meristem identity gene product ligand-dependent such that, upon contacting the transgenic seed plant with cognate ligand, floral meristem identity gene product activity is increased.

Methods for constructing a nucleic acid molecule encoding a chimeric protein of the invention are routine and well known in the art (Sambrook et al., supra, 1989). Methods of constructing, for example, a nucleic acid encoding an AP1-glucocorticoid receptor WO 97/46078 PCTIUS96/09453 66 ligand binding domain chimeric protein are described in Example IV. For example, the skilled artisan recognizes that a stop codon encoded by the nucleic acid molecule must be removed and that the two nucleic acid molecules must be linked in frame such that the reading frame of the 3' nucleic acid molecule coding sequence is preserved. Methods of transforming a seed plant such as an angiosperm or gymnosperm with a nucleic acid molecule are disclosed above and well known in the art (see Examples I, II and III; see, also, Mohoney et al., U.S.

patent number 5,463,174, and Barry et al., U.S. patent number 5,463,175, each of which is incorporated herein by reference) As used herein, the term "linked in frame," when used in reference to two nucleic acid molecules that make up a nucleic acid molecule encoding a chimeric protein, means that the two nucleic acid molecules are linked in the correct reading frame such that, under appropriate conditions, a full-length chimeric protein is expressed. In particular, a 5' nucleic acid molecule, which encodes the amino-terminal portion of the chimeric protein, must be linked to a 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 translated in the correct reading frame. One skilled in the art would recognize that a nucleic acid molecule encoding a chimeric protein of the invention can comprise, for example, a 5' nucleic acid molecule encoding a floral meristem identity gene product linked in frame to a 3' nucleic acid molecule WO 91. 78 PCT7US96/09453 67 encoding a ligand binding domain or can comprise a nucleic acid molecule encoding a ligand binding domain linked in frame to a 3' nucleic acid molecule encoding a floral meristem identity gene product. Preferably, a nucleic acid molecule encoding a chimeric protein of the invention comprises a 5' nucleic acid molecule encoding a floral meristem identity gene product linked in frame to a 3' nucleic acid molecule encoding a ligand binding domain.

In a transgenic angiosperm containing a chimeric protein of the invention, conversion of shoot meristem to floral meristem can be induced by contacting the transgenic angiosperm with a cognate ligand that is absorbed by the angiosperm and binds the chimeric protein within its ligand binding domain.

This provides a way of converting shoot meristem to floral meristem in an angiosperm by introducing into the angiosperm a nucleic acid molecule encoding a chimeric protein to produce a transgenic angiosperm, where, under appropriate conditions, the chimeric protein containing a floral meristem identity gene product fused to a ligand binding domain is expressed; and contacting the transgenic angiosperm with cognate ligand, where, upon binding of the cognate ligand to the ligand binding domain, floral meristem identity gene product activity is increased, thereby converting shoot meristem to floral meristem in the transgenic angiosperm.

This also allows a method of converting shoot meristem to floral meristem in WO 97/46078 PCT/US96/09453 68 an angiosperm by introducing into the angiosperm a nucleic acid molecule encoding a chimeric protein, which comprises a nucleic acid molecule encoding AP1, CAL or LFY linked in frame to a nucleic acid molecule encoding an ecdysone receptor ligand binding domain, to produce a transgenic angiosperm, where, under appropriate conditions, the chimeric protein is expressed; and contacting the transgenic angiosperm with ecdysone ligand, where, upon binding of the ecdysone ligand to the ecdysone receptor ligand binding domain, floral meristem identity gene product activity is increased, thereby converting shoot meristem to floral meristem in the transgenic angiosperm. Similarly, the invention provides, for example, a method of converting shoot meristem to floral meristem in an angiosperm by introducing into the angiosperm a nucleic acid molecule encoding a chimeric protein, which comprises a nucleic acid molecule encoding AP1, CAL or LFY linked in frame to a nucleic acid molecule encoding a glucocorticoid receptor ligand binding domain, to produce a transgenic angiosperm, where, under appropriate conditions, the chimeric protein is expressed; and contacting the transgenic angiosperm with glucocorticoid ligand, where, upon binding of the glucocorticoid ligand to the glucocorticoid receptor ligand binding domain, floral meristem identity gene product activity is increased, thereby converting shoot meristem to floral meristem in the transgenic angiosperm.

In addition, the invention provides a method of promoting early reproductive development in a seed plant WO 97/46078 PCT/US96/09453 69 by introducing into the seed plant a nucleic acid molecule encoding a chimeric protein of the invention to produce a transgenic seed plant, where, under appropriate conditions, the chimeric protein containing a floral meristem identity gene product fused to a ligand binding domain is expressed; and contacting the transgenic seed plant with cognate ligand, where, upon binding of the cognate ligand to the ligand binding domain, floral meristem identity gene product activity is increased, thereby promoting early reproductive development in the transgenic seed plant. The methods of the invention can be practiced with numerous seed plant varieties. The seed plant can be, for example, an angiosperm such as a cereal plant, leguminous plant, hardwood tree or coffee plant, or can be a gymnosperm such as a pine, fir, spruce or redwood tree.

There is provided, for example, a method of promoting early reproductive development in a seed plant by introducing into the seed plant a nucleic acid molecule encoding a chimeric protein, which comprises a nucleic acid molecule encoding a floral meristem identity gene product linked in frame to a nucleic acid molecule encoding an ecdysone receptor ligand binding domain, to produce a transgenic seed plant, where, under appropriate conditions, the chimeric protein is expressed; and contacting the transgenic seed plant with ecdysone ligand, where, upon binding of the ecdysone ligand to the ecdysone receptor ligand binding domain, floral meristem identity gene product activity is increased, thereby promoting early reproductive development in the 70 transgenic seed plant. Similarly, this allows a method of promoting early reproductive development in a seed plant by introducing into the seed plant a nucleic acid molecule encoding a chimeric protein, which comprises a nucleic acid molecule encoding AP1, CAL or LFY linked in frame to a nucleic acid molecule encoding a glucocorticoid receptor ligand binding domain, to produce a transgenic seed plant, where, under appropriate conditions, the chimeric protein is expressed; and contacting the transgenic seed plant with glucocorticoid ligand, where, upon binding of the glucocorticoid ligand to the glucocorticoid receptor ligand binding domain, floral meristem identity gene product activity is increased, thereby promoting early reproductive development in the transgenic seed plant.

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. In the methods of the present invention, a ligand can be used alone or 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 a seed plant. If desired, a transgenic seed plant of the invention can be contacted with a ligand for increasing floral meristem identity gene product activity in combination with an unrelated molecule such as a plant nutrient, pesticide or insecticide. When used in reference to a particular ligand binding domain, the term WO 97/46078 PCT/US96/09453 71 "cognate ligand" means a ligand that, under suitable conditions, specifically binds the particular ligand binding domain.

One skilled in the art readily can determine the optimum concentration of cognate ligand needed to bind a ligand binding domain and increase floral meristem identity gene product activity in a transgenic seed plant of the invention. Generally, a concentration of about 1 nM to 10 AM cognate ligand is useful for increasing floral meristem identity gene product activity in a transgenic seed plant expressing a chimeric protein of the invention. Preferably, a concentration of about 100 nM to 1 AM cognate ligand is useful for increasing floral meristem identity gene product activity in a transgenic seed plant containing a chimeric protein of the invention (see, for example, Christopherson et al., Proc. Natl.

Acad. Sci. USA 89:6314-6318 (1992), which is incorporated herein by reference; also, see Lloyd et al., supra, 1994). For example, a concentration of about 100 nM to 1 AM dexamethasone can be useful for increasing floral meristem identity gene product activity in a transgenic seed plant of the invention containing a nucleic acid molecule encoding a chimeric protein, which comprises a nucleic acid molecule encoding a floral meristem identity gene product, such as AP1 or CAL, linked in frame to a nucleic acid molecule encoding a glucocorticoid receptor ligand binding domain, as described in Example IV.

As discussed above, a transgenic seed plant of the invention, such as a transgenic seed plant expressing WO 97/46078 PCT/US96109453 72 a chimeric protein of the invention, can be contacted in a variety of manners. A transgenic seed plant can be contacted with cognate ligand, for example, by spraying the seed plant with a gaseous ligand or with solution such as an aqueous solution containing the appropriate ligand; or by adding the cognate ligand to the water supply of a seed plant grown using irrigation or grown hydroponically; or by adding the cognate ligand to the soil or other solid nutrient medium in which a seed plant is grown, whereby the cognate ligand is absorbed into the seed plant to increase floral meristem identity gene product activity. A transgenic seed plant expressing a chimeric protein of the invention also can be contacted with a cognate ligand in aerosol form. In addition, a transgenic seed plant can be contacted with cognate ligand by injecting the seed plant or by immersing the seed plant in a solution containing the cognate ligand.

A transgenic seed plant expressing a chimeric protein of the invention can be contacted individually with cognate ligand, or a group of transgenic seed plants can be contacted en masse to increase floral meristem gene product activity synchronously in all seed plants of the group. Furthermore, a variety of means can be used to contact a transgenic seed plant of the invention with cognate ligand to increase floral meristem identity gene product activity. A transgenic seed plant can be contacted with cognate ligand using, for example, a hand held spraying apparatus; conventional yard "sprinkler"; mechanical spraying system, such as an overhead spraying system in a green house; traditional farm machinery, or 73 "crop dusting." As discussed above in regard to the application of inducing agents, the methods of the invention can be practiced using these and other manual or mechanical means to contact a transgenic seed plant with single or multiple applications of cognate ligand.

The nucleic acid molecules encoding floral meristem identity gene products provided herein also can be useful in generating sterile transgenic seed plants and in methods of producing reproductive sterility in seed plants. The methods of the invention involve cosuppression metholodology, where a nucleic acid molecule in the sense orientation is introduced into a seed plant to suppress expression of a homologous endogenous gent, or involve antisense metholodology. This allows for cosuppression and antisense methods of producing reproductively sterile transgenic seed plants as well as the two types of sterile transgenic seed plants produced by these methods.

A method for producing a reproductively sterile transgenic seed plant has a variety of uses including safely growing transgenic trees in close contact with interfertile wild trees, increasing wood production and reducing allergenic pollen production. A method for p producing reproductive sterility in seed plants, which is useful for transgene containment, can allow, for example, the introduction of transgenic trees into the environment.

Of particular concern to the introduction of transgenic trees into the environment is the possibility of enhanced "weediness" or WO 97/46078 PCT/US96/09453 74 the movement of transgenes by cross-fertilization into gene pools of wild relatives. Most commercially grown forest trees, for example, are grown in close proximity to interfertile wild populations, and gene flow within and among tree populations usually is extensive, making the probability of transgene escape from plantations of fertile transgenic trees high. Regulatory agencies have based approval of transgenic tree planting on sexual isolation of the transgenic species; for example, approval of two field tests for transgenic poplars by the Animal and Plant Health Inspection Service (APHIS) was contingent on the trees not being allowed to flower (see, for example, Strauss et al., Molec. Breed 1:5-26 (1995), which is incorporated herein by reference). Thus, transgene containment through, for example, the use of sterile transgenic trees is central to the usefulness of improved transgenic varieties.

Methods of producing reproductively sterile seed plants also can be useful for increasing wood production, since substantial energy and nutrients are committed to reproductive development in trees. For example, in trees such as radiata pine, white spruce, balsam fir and Douglas fir, reduced growth, as measured by height or stem volume, is correlated with the early production of cones (Strauss et al., supra, 1995). Thus, the methods of the invention, which prevent flowering or cone development, for example, by producing reproductive sterility, are useful for growing substantially larger trees, thus increasing wood production.

WO 97/46078 PCT/US96/09453 A method for producing reproductively sterile seed plants also can be useful for alleviating allergies caused by tree pollen. For example, in Japan many people suffer from allergies caused by the most commonly planted forest tree, the conifer sugi (Strauss et al., supra, 1995). The methods of the invention, therefore, can be advantageous for preventing pollen formation in seed plants such as the conifer sugi.

Cosuppression, which relies on expression of a nucleic acid molecule in the sense orientation, is a well known methodology that produces coordinate silencing of the introduced nucleic acid molecule and the homologous endogenous gene (see, for example, Flavell, Proc. Natl.

Acad. Sci.. USA 91:3490-3496 (1994), which is incorporated herein by reference; Kooter and Mol, supra, 1993). Although the mechanism of cosuppression is unknown, cosuppression is induced most strongly by a large number of transgene copies or by overexpression of transgene RNA; cosuppression also can be enhanced by modification of the transgene such that it fails to be translated. Cosuppression has been used successfully to produce sterile plants; for example, a sense nucleic acid molecule containing a full-length fbpl coding sequence under control of the strong CaMV 35S promoter has been introduced into petunia. Two of twenty-one transformants exhibited an abnormal phenotype and contained multiple copies of the fbpl transgene. Furthermore, fbpl expression was undetectable in these sterile transgenic plants, indicating that expression of endogenous fbpl was WO 97/46078 PCT/US96/09453 76 suppressed (Angenent et al., The Plant Journal 4:101-112 (1993), which is incorporated herein by reference).

Antisense nucleic acid molecules, which can act by reducing mRNA translation or by increasing mRNA degradation, for example, also can suppress gene expression of diverse genes and seed plant species (see, for example, Kooter and Mol, Current Opin. Biol.

4:166-171 (1993), which is incorporated herein by reference; see also Strauss et al., supra, 1995) Antisense nucleic acid molecules previously have been used to successfully suppress the expression of a homologous endogenous gene, thereby generating sterile plants. For example, an antisense chalcone synthase gene under control of the CaMV 35S promoter with an anther-specific enhancer sequence effectively suppressed endogenous chalcone synthase expression levels, resulting in male sterility in transgenic petunia plants (van der Meer et al., The Plant Cell Vol 4:253-262 (1992), which is incorporated herein by reference). Similarly, the full-length tomato TM5 MADS box gene, when placed in antisense orientation under control of the CaMV promoter, was used to produce sterile transgenic tomato plants (Pnuell et al., The Plant Cell Vol. 6, 175-186 (1994), which is incorporated herein by reference) Antisense nucleic acid molecules encoding floral meristem identity gene products similarly can be used to produce reproductive sterility in seed plants; however, by preventing reproductive development at the earliest stage, the methods of the invention result in an advantageous energy savings.

77 This allows for a sterile transgenic seed plant such as an angiosperm or gymnosperm containing one or more sense or antisense nucleic acid molecules encoding a floral meristem identity gene product, or a fragment thereof, such that expression of AP1 and LFY gene products, including expression of endogenous API and LFY gene products, is suppressed in the transgenic seed plant.

This also allows for example, a sterile transgenic seed plant containing a sense or antisense nucleic acid molecule encoding API, or a fragment thereof; a sense or antisense nucleic acid molecule encoding CAL, or a fragment thereof; and a sense or antisense nucleic acid molecule encoding LFY, or a fragment thereof, such that expression of API and LFY gene products, including expression of endogenous AP1 and WY gene products, is suppressed in the transgenic seed plant. This further allows for a sterile tranagenic seed plant containing a sense or antisense nucleic acid molecule encoding AP1, or a fragment thereof, and a sense or antisense nucleic acid molecule encoding WY, or a fragment thereof, such that expression of API and WY gene products, including expression of endogenous AP1 and LFY gene products, is suppressed in the transgenic seed plant.

This also allows for methods of producing reproductive sterility in a seed plant such as a tree by introducing into a seed plant one or more sense or antisense nucleic acid molecules encoding a floral meristem identity gene product, or a fragment thereof, to produce a transgenic seed plant, such that expression of 78 AP1 and LFY gene products, including expression of endogenous AP1 and LFY gene products, is suppressed in the transgenic seed plant. Preferably, there are provided methods of producing reproductive sterility in a seed plant by introducing into a seed plant a sense or antisense nucleic acid molecule encoding AP1, or a fragment thereof; a sense or antisense nucleic acid molecule encoding CAL, or a fragment thereof; and a sense or antisense nucleic acid molecule encoding LFY, or a fragment thereof, to produce a transgenic seed plant, such that expression of API and LFY gene products, including expression of endogenous API and WY gene products, is suppressed in the transgenic seed plant. But alternatively there is provided methods of producing reproductive sterility in a seed plant by introducing into a seed plant a sense or antisense nucleic acid molecule encoding AP1, or a fragment thereof, and a sense or antisense nucleic acid molecule encoding LFY, or a fragment thereof, to produce a transgenic seed plant, such that expression of AP1 and WY gene products, including expression of endogenous AP1 and LFY gene products, is suppressed in the transgenic seed plant.

Sterile seed plants that lack expression of functional API and LFY gene products have been described previously. For example, a non-flowering Arabidopsis Ify apl double mutant has been described in which flowers were transformed into shoot-like structures (see, for example, Bowman et al., supra, 1993, and Weigel, supra,30 1995).

However, in contrast to previously described WO 97, .A78 PCTIUS96/09453 79 methods of generating sterile seed plants using mutagenesis, a methodology that is cumbersome or unfeasible in higher plants, this technique provides a convenient method of producing reproductive sterility in a seed plant using sense or antisense nucleic acid molecules encoding floral meristem identity gene products.

The methods described above for producing reproductive sterility rely upon introducing into a seed plant one or more sense or antisense nucleic acid molecules encoding a floral meristem identity gene product, or a fragment thereof, such that expression of API and LFY gene products, including expression of endogenous AP1 and LFY gene products, is suppressed in the transgenic seed plant. The skilled artisan will recognize that effective suppression of endogenous AP1 and LFY gene product expression depends upon the one or more introduced nucleic acid molecules having a high percentage of homology with the corresponding endogenous gene loci.

The homology requirement for effective suppression using sense or antisense nucleic acid molecules can be determined empirically. In general, a minimum of about 80-90% nucleic acid sequence identity is preferred for effective suppression of endogenous floral meristem identity gene product expression. Thus, a nucleic acid molecule encoding a gene ortholog from the family or genus of the seed plant species into which the nucleic acid molecule is to be introduced is preferable WO 97/46078 PCT, )6109453 in practicing the methods of the invention. More preferably, a nucleic acid molecule encoding a gene ortholog from the same seed plant species into which the nucleic acid molecule is to be introduced is used in the methods of the invention. Although a highly homologous nucleic acid molecule is preferred in the methods of the invention, the sense or antisense nucleic acid molecule need not contain the entire coding sequence of the floral meristem identity gene sequence to be suppressed. Thus, sense or antisense nucleic acid molecule encoding only a fragment of API, CAL or LFY coding sequence, for example, also can be useful in the methods described above.

As used herein in reference to a nucleic acid molecule encoding a floral meristem identity gene product, the terms "sense" and "antisense" have their commonly understood meanings.

As used herein in reference to a nucleic acid molecule encoding a floral meristem identity gene product, the term "fragment" means a portion of the nucleic acid sequence containing at least about 50 base pairs to the full-length of the nucleic acid molecule encoding the floral meristem identity gene product. In contrast to an active fragment, as defined herein, a fragment of a nucleic acid molecule encoding a floral meristem identity gene product need not encode a functional portion of a gene product.

WO 97, .078 PCT/US96/09453 81 The methods for producing reproductive sterility, the sense or antisense nucleic acid molecule is expressed under control of a strong promoter that is expressed, at least in part, in floral meristem. The constitutive cauliflower mosaic virus promoter (Odell et al., supra, 1985), for example, or other strong promoters as disclosed herein, can be useful in the-methods of the invention. In addition, an RNA polymerase III promoter can be useful in methods of producing reproductive sterility using an antisense nucleic acid molecule (see, for example, Bourque and Folk, Plant Mol. Biol. 19':641-647 (1992), which is incorporated herein by reference).

The present invention provides novel substantially purified nucleic acid molecules encoding floral meristem identity gene products. The invention provides a substantially purified nucleic acid molecule encoding Brassica oleracea AP1 having the amino acid sequence SEQ ID NO: 4; a substantially purified nucleic acid molecule encoding Brassica oleracea var. botrytis AP1 having the amino acid sequence SEQ ID NO: 6; or a substantially purified nucleic acid molecule encoding Zea mays AP1 having the amino acid sequence SEQ ID NO: 8. In addition, the invention provides a substantially purified nucleic acid molecule that encodes a Brassica oleracea AP1, Brassica oleracea var. botrytis AP1 or Zea mays AP1 and that contains additional 5' or 3' noncoding sequence.

For example, a substantially purified nucleic acid molecule having a nucleotide sequence such as SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7 is provided.

82 As used herein in reference to a particular nucleic acid molecule or gene product, the term "substantially purified" means that the particular nucleic acid molecule or gene product is in a form that 5 is relatively free from contaminating lipids, unrelated gene products, unrelated nucleic acids or other cellular material normally associated with the particular nucleic acid molecule or gene product in a cell.

The present invention also provides a nucleotide sequence having at least ten contiguous nucleotides of a nucleic acid molecule encoding Brassica oleracea API, Brassica oleracea var. botrytis API or Zea mays AP1, provided that said nucleotide sequence does not encode a portion of a MADS domain. In particular, such a nucleotide sequence can have at least ten contiguous nucleotides of a nucleic acid molecule encoding an AP1 gene product having the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8. A nucleotide sequence of the invention can have, for example, at least ten contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7.

As used herein, the term "contiguous," as used in reference to the nucleotides of a nucleic acid molecule means that the nucleotides of the nucleic acid molecule follow continuously in sequence. Thus, a nucleotide sequence of the invention has at least ten contiguous nucleotides of one of the recited nucleic acid molecules without any extraneous intervening nucleotides.

WO 97/46078 PCT/US96/09453 83 Explicitly excluded from a nucleotide sequence of the present invention is a nucleotide sequence having at least ten contiguous nucleotides that is present in a nucleic acid molecule encoding a MADS domain containing protein. MADS domain containing proteins are well known in the art as described in Purugganan et al., supra, 1995.

In general, a nucleotide sequence of the invention can range in size from about 10 nucleotides to the full-length of a cDNA. Such a nucleotide sequence can be chemically synthesized, using routine methods or can be purchased from a commercial source. In addition, such a nucleotide sequence can be obtained by enzymatic methods such as random priming methods, polymerase chain reaction (PCR) methods or by standard restriction endonuclease digestion, followed by denaturation (Sambrook et al., supra, 1989).

A nucleotide sequence of the invention can be useful, for example, as a primer for PCR (Innis et al.

PCR Protocols: A Guide to Methods and Applications, San Diego, CA: Academic Press, Inc. (1990)). Such a nucleotide sequence generally contains from about 10 to about 50 nucleotides.

A nucleotide sequence of the invention also can be useful in screening 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 having at least ten WO 97/46078 PCT/US96/09453 84 contiguous nucleotides of the nucleic acid molecule encoding Zea mays API (SEQ ID NO: 7) in order to isolate a rice or wheat ortholog of API. Generally, a nucleotide sequence useful for screening a cDNA or genomic library contains at least about 14 to 16 contiguous nucleotides depending, for example, on the hybridization conditions to be used. A nucleotide sequence containing at least 18 to 20 nucleotides, or containing at least 21 to nucleotides, also can be useful.

A nucleotide sequence having at least ten contiguous nucleotides of a nucleic acid molecule encoding Zea mays AP1 (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. Similarly, a nucleotide sequence having at least ten contiguous nucleotides of a nucleic acid molecule encoding Brassica oleracea AP1 (SEQ ID NO: 3) or a nucleotide sequence having at least ten contiguous nucleotides of a nucleic acid molecule encoding Brassica oleracea var. botrytis AP1 (SEQ ID NO: 5) can be used to screen a Brassica oleracea or Brassica oleracea var. botrytis cDNA library to isolate a novel sequence that is related to but distinct from API. In addition, a nucleotide sequence of the invention can be useful in analyzing 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.

WO 97/46078 PCT/US96/09453 The invention also provides a vector containing a nucleic acid molecule encoding a Brassica oleracea AP1 gene product, Brassica oleracea var. botrytis API gene product or Zea mays API 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, supra, 1993).

The invention further provides a method of producing an AP1 gene product by expressing a nucleic acid molecule encoding an AP1 gene product. Thus, a Brassica oleracea AP1 gene product can be produced according to a method of the invention by expressing a nucleic acid molecule having the amino acid sequence of SEQ ID NO: 4 or by expressing a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 3.

Similarly, a Brassica oleracea var. botrytis AP1 gene product can be produced according to a method of the invention by expressing a nucleic acid molecule having the amino acid sequence of SEQ ID NO: 6 or by expressing a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 5. A Zea mays AP1 gene product can be produced by expressing a nucleic acid molecule having the amino acid sequence of SEQ ID NO: 8 or by expressing a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 7.

WO 97/46078 PCT/US96/09453 86 The invention also provides a substantially purified API gene product, such as a substantially purified Brassica oleracea AP1 gene product having amino acid sequence SEQ ID NO: 4; a substantially purified Brassica oleracea var. botrytis API gene product having amino acid sequence SEQ ID NO: 6; or a substantially purified Zea mays AP1 gene product having amino acid sequence SEQ ID NO: 8. As used herein, the term "gene product" is used in its broadest sense and includes 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 "gene product," "protein" and "polypeptide" are used interchangeably. 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 dozen amino acids. The term gene product as used herein includes any such amino acid sequence.

An active fragment of a floral meristem identity gene product also can be useful in the methods of the invention. As 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 in an angiosperm. An active fragment of an AP1 gene product can consist, for example, of an amino acid sequence that is derived from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8 and has WO 97/46078 PCT/US96/09453 87 activity in converting shoot meristem to floral meristem in an angiosperm. An active fragment can be, for example, an amino terminal, carboxyl terminal or internal fragment of Zea mays AP1 (SEQ ID NO: 8) that has activity in converting shoot meristem to floral meristem in an angiosperm. The skilled artisan will recognize that an active fragment of a floral meristem identity gene product, as defined herein, can be useful in the methods of the invention for converting shoot meristem to floral meristem in an angiosperm, for producing early reproductive development in a seed plant, or for producing reproductive sterility in a seed plant.

Such an active fragment can be produced using well known recombinant DNA methods (Sambrook et al., supra, 1989). Similarly, an active fragment can be, for example, an amino terminal, carboxyl terminal or internal fragment of Arabidopsis thaliana CAL (SEQ ID NO: 10) or Brassica oleracea CAL (SEQ ID NO: 12) that has activity, for example, in converting shoot meristem to floral meristem in an angiosperm. The product of the BobCAL gene (SEQ ID NO: 24), 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" of a floral meristem identity gene product.

An active fragment of a floral meristem identity gene product, which can convert shoot meristem to floral meristem in an angiosperm, can be identified WO 97/46078 PCT/US96/09453 88 using the methods described in Examples I, II and III.

Briefly, an angiosperm such as 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 portion can convert shoot meristem to floral meristem and, therefore, is an active fragment of a floral meristem identity gene product.

The invention further provides an antibody that specifically binds an AP1 gene product having the amino acid sequence of Brassica oleracea AP1 (SEQ ID NO: 4); the amino acid sequence of Brassica oleracea var.

botrytis API (SEQ ID NO: or the amino acid sequence of Zea mays AP1 (SEQ ID NO: As used herein, the term "antibody" is used in its broadest sense to include naturally occurring and non-naturally occurring polyclonal and monoclonal antibodies, as well as a polypeptide fragment of an antibody that retains a specific binding activity of at least about 1 x 10 5

M-

1 and preferably about 1 x 106 M- 1 for an API gene product having amino acid sequence SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8. One skilled in the art would know that an antibody fragment such as a Fab, F(ab') 2 or Fv fragment can retain specific binding activity for an AP1 gene product and, thus, is included within the definition of an antibody. A non-naturally occurring antibody, or fragment thereof, such as a chimeric antibody or humanized antibody also is included within the meaning of the term antibody. Such a non-naturally occurring antibody can be constructed using solid phase peptide synthesis, produced recombinantly or obtained, for WO 97/46078 PCT/US96/09453 89 example, by screening a combinatorial library consisting of variable heavy chains and variable light chains as described by Huse et al., Science 246:1275-1281 (1989), which is incorporated herein by reference.

An antibody "specific for" a gene product, or that "specifically binds" a gene product, binds with substantially higher affinity to that gene product than to an unrelated gene product. An antibody specific for a gene product also can have specificity for a related gene product. For example, an antibody specific for a Zea mays AP1 gene product also can specifically bind an Arabidopsis thaliana AP1 gene product or a Brassica oleracea AP1 gene product.

An antibody that specifically binds a Zea mays AP1 gene product (SEQ ID NO: for example, can be prepared using a Zea mays AP1 fusion protein or a synthetic peptide encoding a portion of Zea mays AP1 (SEQ ID NO: 8) as an immunogen. One skilled in the art would know that purified Zea mays AP1 gene product, which can be prepared from a natural source or produced recombinantly according to a method of the invention, or a fragment of a Zea mays AP1 gene product, including a peptide portion of Zea mays AP1 such as a synthetic peptide, can be used as an immunogen. For example, preparation of antisera that specifically binds an AP1 gene product is described in Example VI using a GST-AP1 fusion protein containing amino acids 190 to 251 of AP1 as an immunogen. In addition, a non-immunogenic fragment or synthetic peptide derived from Zea mays AP1, for 90 example, can be made immunogenic by coupling the nonimmunogenic fragment or peptide (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 as described, for example, by Harlow and Lane, Antibodies* A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1988), which is incorporated herein by reference.

The invention also provides an expression vector comprising a nucleic acid molecule encoding a AP1 product having an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:6 SEQ ID NO: 8.

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.

As used herein, the term "heterologous regulatory element" means a regulatory element derived from a different gene than the gene encoding the floral meristem identity gene product to which it is operably linked. A vector containing a floral meristem identity gene, however, contains a nucleic acid molecule encoding a floral meristem identity gene product operably linked to a homolgous regulatory element. Such a vector does WO 97/46078 PCT/US96/09453 91 not contain a nucleic acid molecule encoding a floral meristem identity gene product operably linked to a heterologous regulatory element and, thus, is not an expression vector of the invention.

The invention further provides a plant expression vector containing a floral meristem identity gene product operably linked to a heterologous regulatory element. For example, a plant expression vector containing a nucleic acid molecule encoding an APl gene product having at least about 70 percent amino acid identity with an amino acid sequence of Arabidopsis thaliana APl (SEQ ID NO: 2) in the region from amino acid 1 to amino acid 163 or with the amino acid sequence of Zea mays API (SEQ ID NO: 8) in the region from amino acid 1 to amino acid 163 is provided. A plant expression vector containing a floral meristem identity gene product operably linked to a constitutive regulatory element, such as the cauliflower mosaic virus 35S promoter, is provided. In addition, a plant expression vector containing a floral meristem identity gene product operably linked to an inducible regulatory element is provided.

A useful plant expression vector can contain a constitutive regulatory element for expression of an exogenous nucleic acid molecule in all or most tissues of a seed plant. The use of a constitutive regulatory element can be particularly advantageous because expression from the element is relatively independent of developmentally regulated or tissue-specific factors.

WO 97/46078 PCT/US96/09453 92 For example, the cauliflower mosaic virus 35S promoter (CaMV 35S) is a well-characterized constitutive regulatory element that produces a high level of expression in all plant tissues (Odell et al., Nature 313:810-812 (1985), which is incorporated herein by reference). Furthermore, the CaMV 35S promoter can be particularly useful due to its activity in numerous different seed plant species (Benfey and Chua, Science 250:959-966 (1990), which is incorporated herein by reference; Odell et al., supra, 1985). Other constitutive regulatory elements useful for expression in a seed plant include, for example, the cauliflower mosaic virus 19S promoter; the Figwort mosaic virus promoter (Singer et al., Plant Mol. Biol. 14:433 (1990), which is incorporated herein by reference); and the nopaline synthase (nos) gene promoter (An, Plant Physiol. 81:86 (1986), which is incorporated herein by reference).

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), which is incorporated herein by reference), or the AGL8 promoter, which is active in the apical shoot meristem immediately after the transition to flowering (Mandel and Yanofsky, supra, 1995). The promoter of the SHOOTMERISTEMLESS gene, which is expressed exclusively in the shoot meristem beginning within an embryo and throughout the angiosperm life cycle, also can be a particularly useful

I?

WO 97/46078 PCTIUS96/09453 93 tissue-specific gene regulatory element (see Long et al., Nature 379:66-69 (1996), which is incorporated herein by reference) An appropriate regulatory element such as a promoter 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 if expression of a gene product in all plant tissues is desired. In addition, a developmentally regulated or tissue-specific regulatory element can be useful to direct floral meristem identity gene expression to specific tissues, for example. As discussed above, inducible expression also can be particularly useful to manipulate the timing of gene expression such that, for example, a population of transgenic seed plants of the invention that contain an expression vector comprising a floral meristem identity gene linked to an inducible regulatory element can undergo early reproductive development at essentially the same time. Selecting the time of reproductive development can be useful, for example, in manipulating the time of crop harvest.

Using nucleic acid molecules encoding API provided herein, the skilled artisan can isolate, if desired, a novel ortholog of APi. For example, one would choose a region of API that is highly conserved among known API sequences such as a region that is highly conserved between Arabidopsis API (SEQ ID NO: 1) and Zea mays API (GenBank accession number L46400; SEQ ID NO: 7) WO 97/46078 PCTIUS96/09453 94 to screen a cDNA or genomic library of interest for a novel AP1 ortholog. One can use a full-length Arabidopsis API (SEQ ID NO: for example, to isolate a novel ortholog of API (see Example If desired, the region encoding the MADS domain, which is common to a number of genes, can be excluded, from the sequence used as a probe. Similarly, the skilled artisan knows that a nucleic acid molecule encoding a full-length CAL cDNA such as Arabidopsis CAL (SEQ ID NO: 9) or Brassica oleracea CAL (SEQ ID NO: 11) can be useful in isolating a novel CAL ortholog.

For example, the Arabidopsis API cDNA (SEQ ID NO: 1) can be used as a probe to identify and isolate a novel API ortholog. Using a nucleotide sequence derived from a conserved region of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7, for example, a nucleic acid molecule encoding a novel API ortholog can be isolated from other plant species. Using methods such as those described by Purugganan et al., supra, 1995, one can readily confirm that the newly isolated molecule is an API ortholog. Thus, a nucleic acid molecule encoding an AP1 gene product, which has at least about 70 percent amino acid identity with the amino acid sequence of SEQ ID NO: 2 (Arabidopsis API) in the region from amino acid 1 to amino acid 163 or with the amino acid sequence of SEQ ID NO: 8 (Zea mays AP1) in the region from amino acid 1 to amino acid 163 can be isolated and identified using well known methods.

WO 97/46078 PCT/US96/09453 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 a region conserved between Arabidopsis CAL (SEQ ID NO: 9) and Brassica oleracea CAL (SEQ ID NO: 11). The Arabidopsis CAL cDNA (SEQ ID NO: 9) or Brassica oleracea CAL cDNA (SEQ ID NO: 11), or a nucleotide fragment thereof, can be used to identify and isolate a novel CAL ortholog using methods such as those described in Example V. In order to identify related MADS domain genes, a nucleotide sequence derived from the MADS domain of API or CAL, for example, can be useful to isolate a related gene sequence encoding this DNA-binding motif.

Hybridization conditions for isolating a gene ortholog, for example, are relatively stringent 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 kit for converting shoot meristem to floral meristem in an angiosperm, which contains a plant expression vector having a nucleic acid molecule encoding a floral meristem identity gene product. A kit for promoting early reproductive development in a seed plant, which contains WO 97/46078 PCT/US96/09453 96 a plant expression vector having a nucleic acid molecule encoding a floral meristem identity gene product, also is provided. If desired, such kits can contain appropriate reagents to facilitate high efficiency transformation of a seed plant with a plant expression vector of the invention. Furthermore, if desired, a control vector lacking a floral meristem identity gene can be included in the kits to determine, for example, the efficiency of transformation.

The following examples are intended to illustrate but not limit the present invention.

EXAMPLE I Conversion of shoot meristem to floral meristem and early reproductive development in an APETALA1 transqenic plant This example describes methods for producing a transgenic Arabidopsis plant containing ectopically expressed AP1.

A. Ectopic expression of APETALAl converts inflorescence shoots into flowers Transgenic plants that constitutively express API from the cauliflower mosaic virus 35S (CaMV promoter were produced to determine whether ectopic API expression was sufficient to convert shoot meristem to floral meristem. The API coding sequence was placed under control of the CaMV 35S promoter (Odell et al., WO97/46078 PCT/US96/09453 97 supra, 1985) as follows. Bam HI linkers were ligated to the Hinc II site of the full-length API complementary DNA (Mandel et al., supra, (1992), which is incorporated herein by reference) in pAM116, and the resulting Bam HI fragment was fused to the CaMV 35S promoter (Jack et al., Cell 76:703-716 (1994), which is incorporated herein by reference) in pCGN18 to create pAM563.

Transgenic 35S-API Arabidopsis plants of the Columbia ecotype were generated by selecting kanamycin-resistant plants after Agrobacterium-mediated plant transformation using the in planta method (Bechtold et al., C.R. Acad. Sci. Paris 316:1194-1199 (1993), which is incorporated herein by reference). All analyses were performed in subsequent generations. Approximately 120 independent transgenic lines that displayed the described phenotypes were obtained.

Remarkably, in 35S-API transgenic plants, the normally indeterminate shoot apex prematurely terminated as a floral meristem and formed a terminal flower.

Generally, 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.

.WO 97/46078 PCT/US96/09453 98 B. LEAFY is not required for the conversion of inflorescence shoots to flowers in an APETALA1 transaenic plant To determine whether the 35S-API transgene causes ectopic LFY activity, and whether ectopic LFY activity is required for the conversion of shoot meristem to floral meristem, the 35S-API transgene was introduced into Arabidopsis Ify mutants. The 35S-API transgene was crossed into the strong lfy-6 mutant background and the F 2 progeny were analyzed.

Mutant ify plants containing the 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 resulting flowers had the typical Ify 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 meristem in a transgenic angiosperm that ectopically expresses API.

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 in specifying sepal and petal identity at a later stage in flower development. Although API RNA initially is expressed throughout the young flower primordium, it is WO 97/46078 PCT/US96/09453 99 later excluded from stamen and carpel primordia (Mandel et al., supra, 1992). Since the CaMV 35S promoter is active in all floral organs, 35S-AP1 transgenic plants are likely to ectopically express AP1 in stamens and carpels. However, the normal stamens and carpels 35S-AP1 in transgenic plants indicate that AP1 is not sufficient to specify sepal and petal organ fate.

D. Ectopic expression of APETALAI causes early reproductive development In addition to its ability to alter inflorescence meristem identity, ectopic expression of API also influences the vegetative phase of plant growth.

Wild-type Arabidopsis 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-API 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 wild-type and 35S-AP1 transgenic plants flowered after producing 52.42±3.47 and 7.4±1.18 leaves, respectively.

Under continuous light growth conditions, flowers appear on wild-type Arabidopsis plants after approximately 18 days, whereas the 35S-AP1 transgenic WO 97/46078 PCTIUS96/09453 100 plants flowered after an average of only 10 days.

Furthermore, under short-day growth conditions, flowering is delayed in wild-type Arabidopsis plants until approximately 10 weeks after germination, whereas transgenic plants flowered in less than about five weeks.

Thus, ectopic API expression significantly reduced the time of reproductive development, as indicated by the time of flowering. Ecotopic API expression also reduced the delay of flowering caused by short day growth conditions.

EXAMPLE II Conversion of inflorescence shoots into flowers in an CAULIFLOWER transgenic plant This example describes methods for producing a transgenic Arabidopsis plant that ectopically expresses

CAL.

Transgenic Arabidopsis plants that ectopically express CAL in shoot meristem were generated. The full-length CAL cDNA was inserted downstream of the CaMV 35S promoter in the Eco RI site of pMON530 (Monsanto Co., St. Louis, Missouri) This plasmid was introduced into Agrobacterium strain ASE and used to transform the Columbia ecotype of Arabidopsis using the modified vacuum infiltration method described by Bechtold et al., supra, 1993. The 96 transgenic lines that harbored the construct had a range of weak to strong phenotypes.

Transgenic plants with the strongest phenotypes (27 WO 97/46078 PCT/US96/09453 101 lines) had a phenotype that closely resembled the tfl mutant phenotype.

The apical and lateral inflorescence shoots of transgenic plants were converted into flowers.

Furthermore, the 35S-CAL transgenic plants were characterized by early reproductive development, as indicated by an early flowering phenotype. These results demonstrate that ectopic expression of CAL is sufficient for the conversion of shoots to flowers and for promoting early reproductive development.

EXAMPLE III Conversion of shoots into flowers and early reproductive developemnt in a LEAFY transgenic plant This example describes methods for producing transgenic Arabidopsis ectopically expressing LFY and transgenic aspen ectopically expressing LFY.

A. Conversion of Arabidopsis shoots and early Arabidopsis reproductive development by LEAFY Transgenic Arabidopsis plants were generated by transforming Arabidopsis with LFY under the control of the CaMV 35S promoter (Odell et al., supra, (1985)). A LFY complementary DNA (Weigel et al, Cell 69:843-859 (1992), which is incorporated herein by reference) was inserted into a T-DNA transformation vector containing a CaMV 35S promoter and a 3' nos cassette (Jack et al., WO 97/46078 PCT/US96/09453 102 supra, 1994). Transformed seedlings were selected for kanamycin resistance. Several hundred Arabidopsis 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 35S-LFY transgenics. In general, Nossen lines had weaker phenotypes, especially when grown under short day conditions. The transgene of line DW151.117 (ecotype Wassilewskija) was introgressed into the erecta background by backcrossing to a Landsberg erecta strain. 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 Ify mutant phenotype when crossed into the background of the lfy-6 null allele.

Most 35S-LFY transgenic plants lines demonstrated a very similar, dominant and heritable phenotype. Secondary shoots that arose in lateral positions were consistently replaced by solitary flowers, and higher-order shoots were absent. Although the number of rosette leaves was unchanged from the wild type, plants flowered earlier than wild type: the solitary flowers in the axils of the rosette leaves developed and opened precociously. In addition, the primary shoot terminated with a flower. In transgenics having the most extreme phenotypes, a terminal flower was formed immediately above the rosette.

WO 97/46078 PCT/US96/09453 103 This gain of function phenotype (conversion of shoots to flowers) is the opposite of the Ify loss of function phenotype (conversion of flowers to shoots). These results demonstrate that LFY encodes a developmental switch that is both sufficient and necessary to convert shoot meristem to flower meristem in an angiosperm.

The effects of constitutive LFY expression differ for primary and secondary shoot meristems.

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 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 early reproductive development as indicated by precocious flowering during the vegetative phase of Arabidopsis, the effect of LFY on the flowering of other seed plant species was examined. The perennial tree, hybrid aspen, is derived from parental species that flower naturally only after 8-20 years of growth (Schopmeyer USDA Agriculture Handbook 450: Seeds of Woody Plants in the United States, Washington DC, USA: US Government Printing Office, pp. 645-655 (1974)).

WO 97/46078 PCT/US96/09453 104 transgenic 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. (Transaen. Res. 1:209-220 (1992), which is incorporated herein by reference).

Levels of LFY RNA expression were similar to those of Arabidopsis, as determined by no'rthern 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 in spring, 1995, at Carlshem, Umea, Sweden) from a tree whose age was determined by counting the number of annual rings in a core extracted with an increment borer at meters above ground level. Flowers were fixed in formaldehyde/acetic acid/ethanol and destained in ethanol before photography.

The overall phenotype of 35S-LFY transgenic 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 Arabidopsis and 35S-LFY aspen, solitary flowers were formed instead of shoots in the axils of vegetative WO 97/46078 PCT/US96/09453 105 leaves. Moreover, as in Arabidopsis, the secondary shoots of transgenic 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. Early reproductive development as demonstrated by precocious flowering 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 floral meristem identity gene product can promote early reproductive development in a heterologous angiosperm species.

EXAMPLE IV Dexamethasone-inducible floral meristem identity gene activity in transaenic plants This example describes the construction and characterization of an APl-glucocorticoid receptor ligand binding domain chimera and its dexamethasone-inducible activity in Arabidopsis.

WO 97/46078 PCT/US96/09453 106 A. Construction and characterization of an APi-alucocorticoid receptor ligand bindina domain chimera A nucleic acid molecule encoding an AP1-glucocorticoid receptor ligand binding domain chimera was prepared as follows. Primers corresponding to the translation initiation and termination codons of AP1 were synthesized for PCR amplification of the Arabidopsis AP1 cDNA. Primer 5'-GGATCCGGATCAAAAA~IGGAAGGGGTAG-3' (SEQ ID NO: 25) contains a translation initiation codon, which is indicated by underlining. Primer 5'-GGATCCGCTGCGGCGAAGCAGCCAAGGTTG-3' (SEQ ID NO: 26) contains a modified translation termination site, which is indicated by underlining and allows the nucleic acid molecule encoding AP1 to be linked in frame to the nucleic acid molecule encoding the glucocorticoid receptor (GR) ligand binding domain.

The full length Arabidopsis AP1 cDNA in pAM116 (see Example I) was used as the template for PCR amplification with primers SEQ ID NOS: 25 and 26, each of which contain a Bam H1 site. The resulting Bam HI fragment, which encodes the full-length Arabidopsis API cDNA except for the translation termination codon, was cloned.into the unique Bam HI site of the GR fusion vector constructed by Lloyd et al., supra, 1994. DNA sequence analyses confirmed that the construct contained the predicted nucleotide sequence.

The resulting AP1-GR construct was introduced into Agrobacterium strain ASE, and apl-15 mutant plants WO 97/46078 PCT/US96/09453 107 were transformed using the vacuum infiltration method described in Example I. Approximately 100 independently derived lines were selected in kanamycin for further analysis.

B. Dexamethasone-inducible activity of an APl-glucocorticoid receptor ligand binding domain chimera in Arabidopsis Kanamycin-resistant transgenic Arabidopsis lines are analyzed in subsequent generations for AP1 activity. After application of dexamethasone to transgenic plants, AP1 activity is monitored by visual inspection for 1) flowering that is earlier than wild-type or 2) partial or complete rescue of the apl mutant phenotype.

To assay for dexamethasone-inducible activity, plants are watered with varying concentrations of dexamethasone. A range of dexamethasone concentrations are tested to determine overall levels of AP1 activity and to determine the resulting phenotypes. A concentration of 1 uM or less dexamethasone preferably is used for induction of API activity.

In addition, dexamethasone is applied directly to plants by spraying. Spraying, like watering, leads to a significant induction of AP1 activity, resulting in the corresponding rescue of the apl mutant phenotype and early reproductive development. Although a single application of dexamethasone is sufficient to increase WO 97/46078 PCT/US96/09453 108 AP1 activity and promote early reproductive development, dexamethasone is applied either once, or repeatedly, and the treatments compared for any observed differences under long or short day conditions as disclosed below.

Dexamethasone is applied to plants at various times post-germination. For example, a large number of AP1-GR transgenic Arabidopsis plants are grown, some of which are treated with dexamethasone on day 1 post-germination, some on day 2, etc., all the way up until and beyond the time at which Arabidopsis plants normally flower. These analyses include plants grown under long day, short day, and under a variety of temperatures. For example, Arabidopsis plants, which typically are grown at 25 0 C, also can be analyzed for API activity at 20 0 C and 15 0 C (see, for example, Bowman et al.

Arabidopsis: An Atlas of Morphology and Development, New York: Springer (1994), which is incorporated by reference herein).

EXAMPLE V Identification and characterization of the Zea mays APETALAI cDNA This example describes the isolation and characterization of Zea mays ZAP1 complementary DNA, which is an ortholog of the Arabidopsis floral meristem identity gene API.

WO 97/46078 PCT/US96/09453 109 A. Identification and characterization of a nucleic acid sequence encoding ZAPI The utility of using a cloned floral homeotic gene from Arabidopsis to identify the putative ortholog in maize has been demonstrated previously (Schmidt et al., supra, (1993), which is incorporated herein by reference). As described in Mena et al. (Plant J.

8(6):845-854 (1995)), the maize ortholog of the Arabidopsis API floral meristem identity gene, was isolated by screening a Zea mays ear cDNA library using the Arabidopsis API 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, and screened using the Arabidopsis API cDNA SEQ ID NO: 1 as the probe.

Low-stringency hybridizations with the API probe were conducted as described previously for the isolation of ZAGI using the AG cDNA as a probe (Schmidt et al., supra, 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 Biochemical, Cleveland, Ohio) according to the manufacturer's protocol.

The nucleotide sequence and deduced amino acid sequence of the ZAPI cDNA are provided as SEQ ID NOS: 7 and 8. The deduced amino acid sequence for ZAPI shares 89% identity with Arabidopsis API through the MADS domain (amino acids 1 to 57) and 70% identity through the first 160 amino acids, which includes the K domain. The high level of amino acid sequence identity between ZAPI and WO 97/46078 PCT/US96/09453 110 API (SEQ ID NOS: 8 and as well as the expression pattern of ZAPI in maize florets (see below), indicate that ZAPI is the maize ortholog of Arabidopsis

API.

B. RNA expression pattern of ZAPI Total RNA was isolated from different maize tissues as described by Cone et al., Proc. Natl. Acad.

Sci.. USA 83:9631-9635 (1986), which is incorporated herein by reference. 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 jg) were fractionated on formaldehyde-agarose gels. Gels were stained in a solution of 0.125 gg 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-N® membranes (Amersham International, Arlington Heights, Illinois) according to the manufacturer's WO 97/46078 PCTIUS96/09453 111 instructions. Prehybridization and hybridization solutions were prepared as previously described (Schmidt et al., Science 238:960-963 (1987), which is incorporated herein.by reference). The probe for ZAPI RNA expression studies was a 445 bp Sac I/ Nsi I 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 using hybridization at 60°C in 50% formamide and washes at 65 0

C

in 0.1x SSC and 0.5% SDS.

The strong sequence similarity between ZAPI and API indicated that ZAPI was the ortholog of this Arabidopsis floral meristem identity gene. As a first approximation of whether the pattern of ZAPI expression paralleled that of API, 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: ZAP1 RNA was detected only in male and female inflorescences and in the husk leaves that surround the developing ear. No ZAPI RNA expression was detectable in RNA isolated from root, shoot, leaf, endosperm, or embryo tissue. The restriction of ZAPI expression to terminal and axillary inflorescences is consistent with ZAPI being the Arabidopsis API 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. ZAPI RNA expression was not detected in WO 97/46078 PCTIUS96/09453 112 maize stamens or carpels, whereas high levels of ZAPI RNA were present in developing ear and tassel florets from which stamens and carpels had been removed. Thus, the exclusion of ZAPI expression from 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.

EXAMPLE VI Production and characterization of anti-AP1 antisera This example demonstrates the production and characterization of antisera that specifically binds the Arabidopsis API gene product.

Western blotting was performed with plant tissue extracts and crude antisera from rabbits immunized with a GST-AP1 fusion protein encoding amino acids 190 to 251 of Arabidopsis thaliana AP1 (SEQ ID NO: The C-terminal region of AP1 spanning amino acids 190 to 251 does not include the MADS domain, which is common to a number of proteins. As shown in Figure 1, the anti-AP1 sera reacted with a 90 kDa protein in inflorescence tissue extracts prepared from wild type Arabidopsis thaliana (Landsburg ecotype). As expected, this reactivity was absent from Arabidopsis mutants lacking API such as apl-1 or apl-15 (compare lanes 3 and 4 to lane 2).

WO 97/46078 PCT/US96/09453 113 AP1 expression was reduced significantly in inflorescence tissue extracts from the Arabidopsis ap2-2 mutant as compared to wild type plants, indicating that AP2 normally functions to increase or maintain the level of AP1 RNA or protein expression (see lanes 2 and Similarly, reduced AP1 expression in 1fy-6 mutant plants indicates that LFY normally functions to enhance expression of AP1 at the RNA or protein level (see lanes 2 and In contrast to the results seen in ap2-2 or lfy-6 mutant inflorescences, API protein expression in inflorescence tissue from ag-2 mutants is enhanced strikingly as compared to the level of AP1 protein seen in wild type inflorescences (see lanes 1 and These results indicate that the AGAMOUS gene product (AG) negatively regulates AP1 RNA or protein expression.

Western analysis further demonstrated that AP1 protein expression is specific to inflorescence tissue since AP1 reactivity is absent from leaf tissue prepared from wild type Arabidopsis plants (Columbia ecotype; lane In transgenic plants constitutively expressing API from the CaMV promoter, however, AP1 protein expression readily was detectable in leaf tissue as shown in lane 8. Reactivity of the anti-AP1 antisera in transgenic leaves but not in wild type Arabidopsis leaves confirmed the specificity of the anti-AP1 sera.

Specificity of the anti-AP1 sera also was demonstrated by specific binding of the antisera to AP1 but not to the closely related CAL gene product. For WO 97/46078 PCTIUS96/09453 114 example, inflorescence tissue extract from an apl-1 or mutant plant (lane 3 or 4, respectively), which contains CAL but not AP1, was not reactive with the anti-AP1 rabbit sera. These data indicate that the anti-AP1 sera does not react with the CAL gene product.

For production of anti-AP1 sera, a Sty I fragment of the Arabidopsis thaliana API cDNA, which encodes amino acids 190 to 251, was gel purified, blunt ended with Klenow fragment and ligated into the Sma I site of pGEX3X (Pharmacia, Piscataway, NJ) to make pGEX-AP1 190 o-2 5 for expression of a GST-AP1o 90 2 si fusion protein. DH5a E. coli were transformed with the resulting vector by standard techniques (Sambrook, supra, 1989).

A bacterial culture of a pGEX-APl 19 o- 2 si transformant was grown to an ODo 00 of 0.5, and GST-APl 190 -251 expression was induced by addition of 1mM IPTG. The GST-AP1 190 -2s bacterial pellet was harvested after three hours growth at 37°C, washed once with phosphate-buffered saline (PBS; pH 7.2) and lysed by two cycles of freeze-thawing. The cell lysate was resuspended in one-fiftieth of the culture volume in ice cold EB (2 mM EDTA, 2mM DTT, 1 mM PMSF, 5 Ag/ml leupeptin, 7.5 jg/ml pepstatin, 1% aprotinin in PBS pH 7.2) with 2 mg/ml lysozyme and incubated on ice for 30 minutes. Triton X-100 was added to and the solution was sonicated mildly. The extract was clarified by two successive centrifugations of 1 and 15 minutes, respectively, at 13,000 x g in a microfuge.

WO 97/46078 PCT/US96/09453 115 The GST-APl 1 9 0 -251 fusion protein was purified from the bacterial extract as follows.

Glutathione-Sepharose beads (150 s1), which had been pre-equilibrated in EB with 1% Triton X-100, were added to 1 ml of soluble extract in an Eppendorf tube and incubated on a rotating wheel for 60 minutes at 4 0 C. The beads were washed five times in 1 ml EB with 1% Triton X-100; resuspended in protein sample buffer and loaded on a preparative SDS-PAGE gel (Laemmli, Nature 227:680-685 (1970), which is incorporated herein by reference).

Following electrophoresis, the gel was stained for five minutes in 0.05% Coomassie R250 (Fisher Scientific, Pittsburgh, Pennsylvania) in distilled water and subsequently destained in distilled water. GST-APg1 19 -2s fusion protein was cut out of the gel and electroeluted in 0.5X transfer buffer for 3 hours at 100V as described in Harlow and Lane, supra, 1988. The GST-AP1 1 0 -2 51 fusion protein was emulsified with Freund's adjuvant and injected into rabbits by Immunodynamics (La Jolla, CA).

Crude rabbit serum was used for western analysis at a dilution of 1 to 2000. Binding was detected using a secondary antibody coupled to peroxidase (Promega, Madison, WI; 1 to 2500 dilution) and revealed using an enhanced chemiluminesence kit (Amersham).

Plant protein extracts for western analysis were prepared by homogenizing 100 Al plant tissue with 200 u1 2XFSB (Laemmli, supra, 1970) in a Kontes microfuge tube with a pistil. The extract was denatured in boiling water bath for 5 minutes, sonicated for 1 minute and WO 97/46078 PCT/US96/09453 116 clarified by two successive spins of 5 and 15 minutes in a microfuge at 13'000 x g prior to electophoresis.

EXAMPLE VII Cosuppression of AP1 activity This example demonstrates the use of cosuppression to inhibit endogenous AP1 activity in Arabidopsis.

The full length API cDNA from pAM116 (see Example I) was inserted into the Eco RI site of pMON530, and the resulting construct was introduced into Agrobacterium strain ASE. Wild type Arabidopsis was transformed as described in Example I and analyzed for apl mutant phenotypes. In this way, a large number of independently generated cosuppressed lines were generated. Each of the cosuppressed lines had a phenotype similar or identical to apl-1 mutant plants, which lack AP1 activity, indicating that the activity of both the introduced and endogenous copies of API was suppressed. Analysis of API expression levels by RNA in situ hybridization demonstrated that API expression was reduced and delayed in the cosuppressed transgenic lines having the apl mutant phenotype. Futhermore, in a samll fraction of the cosuppressed transgenic lines, a enhanced phenotype resembling the cauliflower phenotype was observed. This enhanced phenotype indicated that introduction of an API construct can supress expression of both endogenous API and CAL.

WO 97/46078 PCT/US96/09453 117 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 of the invention. Accordingly, the invention is limited only by the following claims.

SWO 97/46078 PCT/US96/09453 118 SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: The Regents of the University of California (ii) TITLE OF INVENTION: Maize and Cauliflower APETALA1 Gene Products and Nucleic Acid Molecules Encoding Same (iii) NUMBER OF SEQUENCES: 26 (iv) CORRESPONDENCE

ADDRESS:

ADDRESSEE: Campbell and Flores STREET: 4370 La Jolla Village Drive, Suite 700 CITY: San Diego STATE: California COUNTRY: USA ZIP: 92122 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE: 05-JUN-1996

CLASSIFICATION:

(viii) ATTORNEY/AGENT

INFORMATION:

NAME: Campbell, Cathryn A.

REGISTRATION NUMBER: 31,815 REFERENCE/DOCKET NUMBER: FP-UD 2142 (ix) TELECOMMUNICATION

INFORMATION:

TELEPHONE: (619) 535-9001 TELEFAX: (619) 535-8949 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 1057 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ix) FEATURE: NAME/KEY: CDS LOCATION: 124..893 (ix) FEATURE: NAME/KEY: miscfeature LOCATION: 1..1057 OTHER INFORMATION: /note= "product Arabidopsis thaliana AP1." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: CTTTCCAATT GGTTCATACC AAAGTCTGAG CTCTTCTTTA TATCTCTCTT GTAGTTTCTT ATTGGGGGTC TTTGTTTTGT TTGGTTCTTT TAGAGTAAGA AGTTTCTTAA AAAAGGATCA 120 WO 97/46078 PCTIUS96/09453 a 11 AAA ATG GGA AGG GGT AGG GTT CAA TTG AAG AC Met Gly Arg Gly Arg Val Gin Leu Lys Ai 1 5 AAT AGA CAA GTG ACA TTC TCG AAA AGA AGA G Asn Arg Gin Val Thr Phe Ser Lys Arg Arg A 25 GCT CAT GAG ATC TCT GTT CTC TGT GAT GCT G Ala His Glu Ile Ser Val Leu Cys Asp Ala G 40 TTC TCC CAT AAG GGG AAA CTC TTC GAA TAC T Phe Ser His Lys Gly Lys Leu Phe Glu Tyr S 55 GAG AAG ATA CTT GAA CGC TAT GAG AGG TAC T Glu Lys Ile Leu Glu Arg Tyr Glu Arg Tyr S 70 CTT ATT GCA CCT GAG TCC GAC GTC AAT ACA A Leu Ile Ala Pro Glu Ser Asp Val Asn Thr A 85 AAC AGG CTT AAG GCT AAG ATT GAG CTT TTG G Asn Arg Leu Lys Ala Lys Ile Glu Leu Leu G 100 105 TAT CTT GGG GAA GAC TTG CAA GCA ATG AGC C Tyr Leu Gly Glu Asp Leu Gin Ala Met Ser 1 115 120 CTG GAG CAG CAG CTT GAC ACT GCT CTT AAG Leu Glu Gin Gin Leu Asp Thr Ala Leu Lys 130 135 AAC CAA CTT ATG TAC GAG TCC ATC AAT GAG Asn Gin Leu Met Tyr Glu Ser Ile Asn Glu 145 150 GCC ATA CAG GAG CAA AAC AGC ATG CTT TCT Ala Ile Gin Glu Gin Asn Ser Met Leu Ser 160 165 GAA AAA ATT CTT AGG GCT CAA CAG GAG CAG Glu Lys Ile Leu Arg Ala Gin Gin Glu Gin 180 185 GGC CAC AAT ATG CCT CCC CCT CTG CCA CCG Gly His Asn Met Pro Pro Pro Leu Pro Pro 195 200 CAT CCT TAC ATG CTC TCT CAT CAG CCA TCT His Pro Tyr Met Leu Ser His Gin Pro Ser 210 215 GGT CTG TAT CAA GAA GAT GAT CCA ATG GCA Gly Leu Tyr Gin Glu Asp Asp Pro Met Ala 225 230 GAA CTG ACT CTT GAA CCC GTT TAC AAC TGC Glu Leu Thr Leu Glu Pro Val Tyr Asn Cys 240 245 GCA TG AAGCATTTCC ATATATATAT TTGTAATCGT Ala 9 G ATA GAG AAC AAG ATC g Ile Glu Asn Lys Ile 0 :T GGT CTT TTG AAG AAA La Gly Leu Leu Lys Lys kA GTT GCT CTT GTT GTC Lu Val Ala Leu Val Val CC ACT GAT TCT TGT ATG er Thr Asp Ser Cys Met CT TAC GCC GAA AGA CAG er Tyr Ala Glu Arg Gin AC TGG TCG ATG GAG TAT sn Trp Ser Met Glu Tyr 90 AG AGA AAC CAG AGG CAT lu Arg Asn Gin Arg His 110 CT AAA GAG CTT CAG AAT Pro Lys Glu Leu Gin Asn 125 AC ATC CGC ACT AGA AAA is Ile Arg Thr Arg Lys 140 CTC CAA AAA AAG GAG AAG Leu Gin Lys Lys Glu Lys 155 AAA CAG ATC AAG GAG AGG Lys Gin Ile Lys Glu Arg 170 175 TGG GAT CAG CAG AAC CAA Trp Asp Gin Gin Asn Gin 190 CAG CAG CAC CAA ATC CAG Gin Gin His Gin Ile Gin 205 CCT TTT CTC AAC ATG GGT Pro Phe Leu Asn Met Gly 220 ATG AGG AGG AAT GAT CTC Met Arg Arg Asn.Asp Leu 235 AAC CTT GGC TGC TTC GCC Asn Leu Gly Cys Phe Ala 250 255 CAACAATAAA AACAGTTTGC 168 216 264 312 360 408 456 504 552 600 648 696 744 792 840 888 943 WO 97/46078 PCTIUS96/09453 120 CACATACATA TAAATAGTGG CTAGGCTCTT TTCATCCAAT TAATATATTT TGGCAAATGT 1003 TCGATGTTCT TATATCATCA TATATAAATT AGCAGGCTCC TTTCTTTTTT TGTA 1057 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 256 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Gly Arg Gly Arg Val Gin Leu Lys Arg Ile Glu Asn Lys Ile Asn 1 5 10 Arg Gin Val Thr Phe Ser Lys Arg Arg Ala Gly Leu Leu Lys Lys Ala 25 His Glu Ile Ser Val Leu Cys Asp Ala Glu Val Ala Leu Val Val Phe 40 Ser His Lys Gly Lys Leu Phe Glu Tyr Ser Thr Asp Ser Cys Met Glu 55 Lys Ile Leu Glu Arg Tyr Glu Arg Tyr Ser Tyr Ala Glu Arg Gin Leu 70 75 Ile Ala Pro Glu Ser Asp Val Asn Thr Asn Trp Ser Met Glu Tyr Asn 90 Arg Leu Lys Ala Lys Ile Glu Leu Leu Glu Arg Asn Gin Arg His Tyr 100 105 110 Leu Gly Glu Asp Leu Gin Ala Met Ser Pro Lys Glu Leu Gin Asn Leu 115 120 125 Glu Gin Gin Leu Asp Thr Ala Leu Lys His Ile Arg Thr Arg Lys Asn 130 135 140 Gin Leu Met Tyr Glu Ser Ile Asn Glu Leu Gin Lys Lys Glu Lys Ala 145 150 155 160 Ile Gin Glu Gin Asn Ser Met Leu Ser Lys Gin Ile Lys Glu Arg Glu 165 170 175 Lys Ile Leu Arg Ala Gln Gin Glu Gin Trp Asp Gin Gin Asn Gin Gly 180 185 190 His Asn Met Pro Pro Pro Leu Pro Pro Gin Gin His Gin Ile Gin His 195 200 205 Pro Tyr Met Leu Ser His Gin Pro Ser Pro Phe Leu Asn Met Gly Gly 210 215 220 Leu Tyr Gin Glu Asp Asp Pro Met Ala Met Arg Arg Asn Asp Leu Glu 225 230 235 240 Leu Thr Leu Glu Pro Val Tyr Asn Cys Asn Leu Gly Cys Phe Ala Ala 245 250 255 WO 97/46078 PCT/US96/09453 121 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 794 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 36..794 (ix) FEATURE: NAME/KEY: misc feature LOCATION: 1..794 OTHER INFORMATION: /note= "product Brassica oleracea AP1." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: TCTTAGAGGA AATAGTTCCT TTAAAAGGGA TAAAA ATG GGA AGG GGT AGG GTT Met Gly Arg Gly Arg Val 1 CAG TTG AAG Gin Leu Lys AAA AGA AGA Lys Arg Arg

AGG

Arg ATA GAA AAC AAG Ile Glu Asn Lys AAT AGA CAA GTG Asn Arg Gin Val ACA TTC TCG Thr Phe Ser TCT GTT CTG Ser Val Leu GCT GGT CTT ATG Ala Gly Leu Met

AAG

Lys AAA GCT CAT GAG Lys Ala His Glu TGT GAT Cys Asp GCT GAA GTT GCG Ala Glu Val Ala GTT GTC TTC TCC Val Val Phe Ser AAG GGG AAA CTC Lys Gly Lys Leu 197

TTT

Phe GAA TAC TCC ACT Glu Tyr Ser Thr TCT TGT ATG Ser Cys Met GAG AAG Glu Lys ATA CTT GAA CGC Ile Leu Glu Arg GAG AGA TAC TCT Glu Arg Tyr Ser GCC GAG AGA CAG Ala Glu Arg Gin

CTT

Leu ATA GCA CCT GAG Ile Ala Pro Glu TCC GAC Ser Asp TCC AAT ACG Ser Asn Thr GAG CTT TTG Glu Leu Leu 105

AAC

Asn TGG TCG ATG GAG Trp Ser Met Glu AAT AGG CTT AAG Asn Arg Leu Lys GCT AAG ATT Ala Lys Ile 100 GAC TTG CAA Asp Leu Gin 341 GAG AGA AAC CAG Glu Arg Asn Gin

AGG

Arg 110 CAC TAT CTT GGG His Tyr Leu Gly

GAA

Glu 115 GCA ATG Ala Met 120 AGC CCT AAG GAA Ser Pro Lys Glu

CTC

Leu 125 CAG AAT CTA GAG CAA CAG CTT GAT ACT 437 Gin Asn Leu Glu Gin Gin Leu Asp Thr 130

GCT

Ala 135 CTT AAG CAC ATC Leu Lys His Ile

CGC

Arg 140 TCT AGA AAA AAC Ser Arg Lys Asn

CAA

Gin 145 CTT ATG TAC GAC Leu Met Tyr Asp

TCC

Ser 150 ATC AAT GAG CTC Ile Asn Glu Leu CAA AGA AAG GAG AAA Gin Arg Lys Glu Lys 155 GCC ATA CAG GAA CAA Ala Ile Gin Glu Gin 160 AAC AGC Asn Ser 165 WO 97/46078 PTU9/95 PCTIUS96/09453 1 ATG CTT TCC AAG CAG ATT AAG GAG AGG GAAA Met Leu Ser, Lys Gin Ile Lys Glu Arg Giu A 170 175 CAA GAG CAA TGG GAC GAG CAG AAC CAT GGCC Gin Giu Gin Trp Asp Giu Gin Asfl His Gly H 185 190 CCA CCC CCG CAG CAG CAT CA.A ATC CAG CAT C Pro Pro Pro Gin Gin His Gin Ile Gin His P 200 205 CAG CCA TCT CCT TTT CTC AAC ATG GGG GGG C Gin Pro Ser Pro Phe Leu Asn Met Gly Giy L 215 220 2 CAA ATG GCA ATG AGG AGG AAC GAT CTC GAT C Gin Met Ala Met Arg Arg Asn Asp Leu Asp I 235 240 TAT AAC TGC AAT CTC GGC TGC Tyr Asn Cys Asn Leu Gly Cys 250 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 253 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Met Gly Arg Gly Arg Val Gin Leu Lys Arg 1 5 Arg Gin Val Thr Phe Ser Lys Arg Arg Ala 25 His Glu Ile Ser Val Leu Cys Asp Ala Giu 40 Ser His Lys Gly Lys Leu Phe Glu Tyr Ser 55 Lys Ilie Leu Giu Arg Tyr Giu Arg Tyr Ser 70 Ile Ala Pro Glu Ser Asp Ser Asn Thr Asn 90 Arg Leu Lys Ala Lys Ile Glu Leu Leu Giu 100 105 Leu Gly Glu Asp Leu Gin Ala Met Ser Pro 115 3.20 Giu Gln Gln Leu Asp Thr Ala Leu Lys His 130 135 Gin Leu Met Tyr Asp Ser Ile Asn Glu Leu 145 150 22

AC

sn

AT

is

CT

ro

TG

~eu 25

TG

~eu

GTT

Val

AAT

Asn

TAC

Tyr 210

TAT

Tyr

TCT

Ser CTT Leu A

I

ATG C Met P 195 ATG C Met L

CAAC

Gln C

CTTC

LeuC Asn Met Leu Ser Giu Met Gin Leu 125 ;Ser 3Lys

.GG

~rg

'CT

~ro

~TC

~eu

;AA

;lu 7AA lu

GCG

Ala

CCG

Pro

TCT

Ser

GAA

Glu

CCC

Pro 245

CAA

Gin

CCT

Pro

CAT

His

GAT

Asp 230

GT

Gly 581 629 677 NO 4: Ile Glu Gly Leu Val Ala Thr Asp Tyr Ala 75 Trp Ser Arg Asn Lys Gli.

Ilie Arc 14 Gin Arc 155 Lys Lys Val Cys Arg Glu Arg 110 Gin Arg Glu Ile Asn Lys Ala Val Phe Met Olu Gin Leu Tyr Asn His Tyr Asn Leu Lys Asn Lys Ala 160 WO 97/46078 PCTIUS96/09453 123 Ile Gin Glu Gin Asn Ser Met Leu Ser Lys Gin Ile Lys Glu Arg Glu 165 170 175 Asn Val Leu Arg Ala Gin Gin Glu Gin Trp Asp Glu Gin Asn His Gly 180 185 190 His Asn Met Pro Pro Pro Pro Pro Pro Gin Gin His Gin Ile Gin His 195 200 205 Pro Tyr Met Leu Ser His Gin Pro Ser Pro Phe Leu Asn Met Gly Gly 210 215 220 Leu Tyr Gin Glu Glu Asp Gin Met Ala Met Arg Arg Asn Asp Leu Asp 225 230 235 240 Leu Ser Leu Glu Pro Gly Tyr Asn Cys Asn Leu Gly Cys 245 250 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 768 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..766 (ix) FEATURE: NAME/KEY: misc_feature LOCATION: 1..768 OTHER INFORMATION: /note= "product Brassica oieracea var. botrytis AP1." (xi) SEQUENCE DESCRIPTION: SEQ ID ATG GGA AGG GGT AGG GTT CAG TTG AAG AGG ATA GAA AAC AAG ATC AAT 48 Met Gly Arg Gly Arg Val Gin Leu Lys Arg Ile Glu Asn Lys Ile Asn 1 5 10 AGA CAA GTG ACA TTC TCG AAA AGA AGA GCT GGT CTT ATG AAG AAA GCT 96 Arg Gin Val Thr Phe Ser Lys Arg Arg Ala Gly Leu Met Lys Lys Ala 25 CAT GAG ATC TCT GTT CTG TGT GAT GCT GAA GTT GCG CTT GTT GTC TTC 144 His Glu Ile Ser Val Leu Cys Asp Ala Glu Val Ala Leu Val Val Phe 40 TCC CAT AAG GGG AAA CTC TTT GAA TAC CCC ACT GAT TCT TGT ATG GAG 192 Ser His Lys Gly Lys Leu Phe Glu Tyr Pro Thr Asp Ser Cys Met Glu 55 GAG ATA CTT GAA CGC TAT GAG AGA TAC TCT TAC GCC GAG AGA CAG CTT 240 Glu Ile Leu Glu Arg Tyr Glu Arg Tyr Ser Tyr Ala Glu Arg Gin Leu 70 75 ATA GCA CCT GAG TCC GAC TCC AAT ACG AAC TGG TCG ATG GAG TAT AAT 288 Ile Ala Pro Glu Ser Asp Ser Asn Thr Asn Trp Ser Met Glu Tyr Asn 90 WO 97/46078 WO 9746078PCT[US96/09453 124

AGG

Arg

CTT

Leu

GAG

Glu

CA-A

Gin 145

ATA

Ile

AAC

Asn

CAT

His

CCT

Pro

CTG

Leu 225

CTG

Leu

GA

CTT

Leu

GGG

Giy

CA-A

Gin 130

CTT

Leu

CAG

Gin

GTT

Val

AAT

A-sn

TAC

Tyr 210

TAT

Tyr

TCT

Ser

A-AG

Lys

GA-A

Giu 115

CAG

Gin

ATG

Met

GA-A

Giu

CTT

Leu

ATG

Met 195

ATG

Met

CA-A

Gin

CTT

Leu GCT A-AG A-TT GAG CTT TTG GAG A-GA A-AC CAG AGG Aila 100

GAC

Asp

CTT

Leu

TAC

Tyr

CAA

Gin

A-GG

A-rg 180

CCT

Pro

CTC

Leu

GA-A

Giu

GAA

Giu Lys

TTG

Leu

GAT

Asp

GAC

Asp

A-AC

Asn 165

GCG

Alia

CCG

Pro

TCT

Ser

GAA

Giu

CCC

Pro 245 Ile

CA-A

Gin

ACT

Thr

TCC

Ser 150

A-GC

Ser

CAA

Gin

CCT

Pro

CAT

His

GAT

Asp 230 Giu

GCA

Ala

GCT

Ala 135

ATC

Ile

ATG

Met

CAA

Gin

CCA

Pro

CAG

Gin 215

CA-A

Gin Leu

ATG

Met 120

CTT

Leu

A-AT

Asn

CTT

Leu

GAG

Giu

CCC

Pro 200

CCA

Pro

ATG

Met Leu 105

AGC

Ser

AAG

Lys

GAG

Glu

TCC

Ser

CAA

Gin 185

CCG

Pro

TCT

Ser

GCA

Ala Giu

CCT

Pro

CAC

His

CTC

Leu

AAG

L.ys 170

TGG

Trp

CAG

Gin

CCT

Pro

A-TG

Met Arg

A-AG

Lys

A-TC

le

CAA

Gin 155

CAG

Gin

GAC

Asp

CAG

Gin

TTT

Phe

AGG

Arg 235 A-sn

GA-A

Giu

CGC

Arg 140

A-GA

Arg

A-TT

Ile

GAG

Giu

CAT

His

CTC

Leu 220

AGG

Arg Gin

CTC

Leu 125

TCT

Ser

A-AG

Lys

A-AG

Lys

CAG

Gin

CAA

Gin 205

AAC

A-sn

A-AC

A-en Arg 110

CAG

Gin

A-GA

Arg

GAG

Giu

GAG

Giu

A-AC

A-sn 190

A-TC

Ile

A-TG

Met

GAT

Asp

CAC

His

A-AT

A sn

AAA

Lys

AA-A

Lys

AGG

Arg 175

CAT

His

CAG

Gin

GGA

Gly

CTC

Leu

TAT

Tyr

CTA

Leu

A-AC

A-sn

GCC

A-ia 160

GA-A

Giu

GGC

Gly

CAT

His

GGG

Giy

GAT

Asp 240 336 384 432 480 528 576 624 672 720 GTT TAC A-AC TGC A-AC CTT GGC! CGT CGC TGC T Val Tyr A-en Cys A-sn Leu Gly Arg A-rg Cys INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 255 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Met Gly Arg Gly A-rg Val Gin Leu Lye Arg Ile Glu A-sn Lye Ile A-sn 1 5 10 A-rg Gin Val Thr Phe Ser Lye Arg Arg A-la Giy Leu Met Lys Lye Ala 25 His Giu Ile Ser Vai Leu Cys Asp A-ia Giu Vai A-ia Leu Val Val Phe 40 Ser His Lye Giy Lye Leu Phe Giu Tyr Pro Thr Asp Ser Cys Met Giu 55 WO 97/46078 PCT/US96/09453 125 Tyr 75 Glu Ile Arg Leu Glu Gin 145 Ile Asn His Pro Leu 225 Leu Ile Ala Leu Gly Gin 130 Leu Gin Val Asn Tyr 210 Tyr Ser Leu Pro Lys Glu 115 Gin Met Glu Leu Met 195 Met Gin Leu Glu Arg Tyr 70 Glu Arg Tyr Ser Ala Glu Arg Gin Glu Ala 100 Asp Leu Tyr Gin Arg 180 Pro Leu Glu Glu Ser Lys Leu Asp Asp Asn 165 Ala Pro Ser Glu Pro 245 Asp Ile Gin Thr Ser 150 Ser Gin Pro His Asp 230 Ser Glu Ala Ala 135 Ile Met Gin Pro Gin 215 Gin Asn Leu Met 120 Leu Asn Leu Glu Pro 200 Pro Met Thr Asn Trp 90 Leu Glu Arg 105 Ser Pro Lys Lys His Ile Glu Leu Gin 155 Ser Lys Gin 170 Gin Trp Asp 185 Pro Gin Gin Ser Pro Phe Ala Met Arg 235 Ser Asn Glu Arg 140 Arg Ile Glu His Leu 220 Arg Met Gin Leu 125 Ser Lys Lys Gin Gin 205 Asn Asn Glu Arg 110 Gin Arg Glu Glu Asn 190 Ile Met Asp Tyr His Asn Lys Lys Arg 175 His Gin Gly Leu Leu Asn Tyr Leu Asn Ala 160 Glu Gly His Gly Asp 240 Val Tyr Asn Cys Leu Gly Arg Arg Cys 255 INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 1345 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 149..968 (ix) FEATURE: NAME/KEY: misc feature LOCATION: 1..1345 OTHER INFORMATION: /note= "product Zea mays API." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: GCACGAGTCC TCCTCCTCCT CGCATCCCAC CCCACCCCAC CTTCTCCTTA AAGCTACCTG CCTACCCGGC GGTTGCGCGC CGCAATCGAT CGACCGGAAG AGAAAGAGCA GCTAGCTAGC TAGCAGATCG GAGCACGGCA ACAAGGCG ATG GGG CGC GGC AAG GTA CAG CTG Met Gly Arg Gly Lys Val Gin Leu 1 PCTfUS96/09453 WO 97/46078 AAG CGG ATA Lys Arg Ile CGG AAC GGC Arg Asn Gly GCC GAG GTC Ala Giu Val TAC GCC ACC Tyr Ala Thr TAT TCC TAT Tyr Ser Tyr GGA AAT TGG Gly Asn Trp ATA CAA AAA Ile Gin Lys 105 AAT CCC AAA Asn Pro Lys AAG CAC ATC Lys His Ile GAG CTA CAC Giu Leu Gir

E

CAG AAG GA; Gin Lys Gb.

170 CAG CAA CA( Gin Gin Gli 185 CAG ACA AG, Gin Thr Se GGA CTG CC Gly Leu Pr GAT AGA GG Asp Arg Gi 23 CCA CTG CC Pro Leu Px 250 CCA TGG Al] Pro Trp Mf 265 GAG AAC AA Giu Asn Ly CTG CTC AJ Leu Leu L GCC GTC A9] Ala Val I] GAC TCC C( Asp Ser A~ GCT GAA A Ala Giu L' TGC CAC G Cys His G TGC CACA Cys His L 1 GAG CTC C Giu Leu G 125 AGA TCA Arg Ser 140 AAG AAGC iLys Lys C k CTT GCG i Leu Ala I CAG CAG a Gin Gin 2 TCA TCA r Ser Ser 205 G CCT CCA o Pro Pro 220 T GAA GAG y Giu Giu 5 'G GGG CAG -o Gly Gin 'G CTG AGC t Leu Ser 10

EC

AG

ys 10

AG

an

LGG

7'AC

'A(

GT1 VJa 19

TC

Se

CA

Hi

CT

Le

GC

Al

C]

2' ATA AAC Ile Asn 15 AAG GCG Lys Ala GTC TTC Val Phe ATG GAC Met Asp GCT CTT Aia Leu 80 TAC AGG Tyr Arg 95 CAC CTG His Leu CAA CTA Gin Leu AAG AGC Lys Ser AGG TCP iArg Sex 160C 2AGG CAC a Arg Gir 175 G CAG TG( 1 Gin Tr 0 G TCC TCI r Ser Se: CAAC AT s Asn Ii 'G GCT GC u Ala Al 24 ~G CAA CC .a Gin Pr 255 ~C CTC A; Ls Leu AE 2.26 CGG CAG GTG ACC TTC TCC AAG CC Arg Gin Val Thr Phe Ser Lys A3 CAC GAG ATC TCC GTC CTC TGC G2 His Glu Ile Ser Vai Leu Cys A~ TCC CCC AAG GGC AAG CTC TAC G Ser Pro Lys Gly Lys Leu Tyr G 50 AAA ATT CTT GAA CGC TAT GAG C' Lys Ile Leu Glu Arg Tyr Giu A 65 ATT TCA GCT GAA TCT GAA AGTG Ile Ser Ala Glu Ser Giu Ser G AAA CTG AAG GCC AAA ATT GAG A Lys Leu Lys Ala Lys Ile Giu T 100 ATG GGA GAG GAT CTA GAG TCT TI Met Gly Glu Asp Leu Glu Sen L 115 1 GAG CAG CAG CTG GAT AGC TCA C Giu Gin Gin Leu Asp Ser Sen I 130 135 *CAC CTT ATG GCC GAG TCT ATT *His Leu Met Ala Giu Se Ile 145 150 CTG CAG GAG GAG AAC AAG GCT Leu Gin Glu Giu Asn Lys Ala 165 AAG, GCC GTC GCG AGC CGG CAG 1Lys Ala Val Ala Sen Ang Gin 180 3 GAC CAG CAG ACA CAT GCC CAG SAsp Gin Gin Thn His Ala Gin 195 CTTC ATG ATG AGG CAG GAT CAG r Phe Met Met Ang Gin Asp Gin 210 215 C TGC TTC CCG CCG TTG ACA ATG e Cys Phe Pro Pro Leu Thr Met 225 230 G GCG GCG GCG GCG CAG CAG CAG a Ala Ala Ala Ala Gin Gin Gin 0 245 G CAG CTC CGC ATC GCA GGT CTG o0 Gli Leu Arg Ile Ala Gly Leu 260 ~GCA T AAGGAGAGGG TCGATGAACA ;n Ala 3p PkG lu rg

AG

iu

CC

hr

TG

4 eu

TG

jeu

VCT

'er

CTG

Leu

CAG

Glin

GCC

Ala 200

CAG

Gin

GGA

Gly

CAG,

Gin

CCA

Pro 220 268 316 364 412 460 508 556 604 652 700 748 796 844 892 940 WO 97/46078 WO 9746078PCTIUS96/09453 127 CATCGACCTC CTCTCTCTCT CTCTCTCGTC ATGGATCATG ACGTACGCGT ACCATATGGT 1048 TGCTGTGCCT GCCCCCATCG ATCGCGAGCA ATGGCACGCT CATGCAAGTG ATCATTGCTC 1108 CCCGTTGGTT AAACCCTAGC CTATGTTCAT GGCGTCAGCA. ACTAAGCTAA ACTATTGTTA 1168 TGTTTGCAAG AAAGGGTAAA CCCGCTAGCT GTGTAATCTT GTCCAGCTAT CAGTATGCTT 1228 GTTACTGCCC AGTTACCCTT GAATCTAGCG GCGCTTTTGG, TGAGAGGGTG CAGTTTACTT 1288 TAAACATGGT TCGTGACTTG CTGTAAATAG TAGTATTAAT CGATTTGGGC ATCTAAA 1345 INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 273 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Met Gly Arg Gly Lys Val Gin Leu Lys Arg Ile Glu Asn Lys Ile Asn 1 5 10 Arg Gin Val Thr Phe Ser Lys Arg Arg Asn Gly Leu Leu Lys Lys Ala 25 His Glu Ile Ser Val Leu Cys Asp Ala Glu Val Ala Val Ile Val Phe 40 Ser Pro Lys Gly Lys Leu Tyr Giu Tyr Ala Thr Asp Ser Arg Met Asp 55 Lys Ile Leu Giu Arg Tyr Glu Arg Tyr Ser Tyr Ala Glu Lys Ala Leu 70 75 Ilie Ser Ala Giu Ser Glu Ser Giu Gly Asn Trp Cys His Glu Tyr Arg 90 Lys Leu Lys Ala Lys Ile Glu Thr Ilie Gin Lys Cys His Lys His Leu 100 105 110 Met Gly Glu Asp Leu Glu Ser Leu Asn Pro Lys Giu Leu Gin Gin Leu 115 120 125 Giu Gin Gin Leu Asp Ser Ser Leu Lys His Ilie Arg Ser Arg Lys Ser 130 135 140 His Leu Met Ala Glu Ser Ile Ser Giu Leu Gin Lys Lys Giu Arg Ser 145 150 155 160 Leu Gin Glu Glu Asn Lys Ala Leu Gin Lys Giu Leu Ala Glu Arg Gin 165 170 175 Lys Ala Val Ala Ser Arg Gin Gin Gin Gin Gin Gin Gin Val Gin Trp, 180 185 190 Asp Gln Gin Thr His Ala Gin Ala Gin Thr Ser Ser Ser Ser Ser Ser 195 200 205 Phe Met Met Arg Gin Asp Gin Gin Gly Leu pro Pro Pro His Asn Ile 210 215 220 WO 97/46078 PCTIUS96/09453 128 Cys Phe Pro Pro Leu Thr Met Gly Asp Arg Gly 225 230 235 Glu Glu Leu Ala Ala 240 Ala Ala Ala Ala Gln 245 Gin Gin Gin Pro Leu 250 Pro Gly Gin Ala Gln Pro 255 Gln Leu Arg Ile Ala Gly Leu Pro Pro Trp 260 265 Met Leu Ser His Leu Asn 270 INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 779 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 10..775 (ix) FEATURE: NAME/KEY: unsure LOCATION: 778..779 OTHER INFORMATION: /note= nucleotides." (ix) FEATURE: NAME/KEY: miscfeature LOCATION: 1..779 OTHER INFORMATION: /note= thaliana CAL." "N one or more "product Arabidopsis (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: TTAAGAGAA ATG GGA AGG GGT AGG GTT GAA TTG AAG AGG ATA GAG AAC Met Gly Arg Gly Arg Val Glu Leu Lys Arg Ile Glu Asn 1 5 AAG ATC Lys Ile AAT AGA CAA Asn Arg Gin GTG ACA TTC TCG AAA AGA Val Thr Phe Ser Lys Arg 20 AGA ACT GGT CTT TTG Arg Thr Gly Leu Leu GCC GAG GTT TCC CTT Ala Glu Val Ser Leu

AAG

Lys AAA GCT CAG GAG Lys Ala Gin Glu

ATC

Ile TCT GTT CTT TGT Ser Val Leu Cys 144 ATT GTC TTC TCC Ile Val Phe Ser CAT AAG GGC AAA TTG TTC GAG TAC TCC TCT GAA TCT 192 His Lys Gly Lys Leu Phe Glu Tyr Ser Ser Glu Ser 55 TGC ATG GAG Cys Met Glu AGA CAG CTG Arg Gin Leu GTA CTA GAA CGC Val Leu Glu Arg GAG AGG TAT Glu Arg Tyr TCT TAC GCC GAG Ser Tyr Ala Glu CAG ACG AAC TGG Gin Thr Asn Trp 240 ATT GCA CCT GAC Ile Ala Pro Asp CAC GTT AAT GCA His Val Asn Ala WO 97/46078 WO 9746078PCTIUS96/09453 129 ATT GAG Ile Giu TCA ATG GAG TAT AGC AGG CTT AAG GCC AAG Ser Met Giu Tyr Ser Arg Leu Lys Ala Lys 100 AAC CAA AGG CAT TAT CTG GGA GAA GAG TTG OTT TTG GAG AGA Leu Leu Giu Arg 105 Asn Gin Arg His Tyr 110 GAT CTC CAA AAT CTG Asp Leu Gin Asn Leu 130 CGC TCC AGA AAA AAT Arg Ser Arg Lys Asn 145 AGA AAG, GAG AAG GAG Arg Lys Giu Lys Giu 160 ATA AAG GAG AGG GAA Ile Lys Glu Arg Giu 175 CAG CTG AAC CGC AGO Gin Leu Asn Arg Ser 190 CAC CCC OAT OTT TAO His Pro His Leu Tyr 210 ATG GGT GGT TTG TAO Met Gly Gly Leu Tyr 225 Leu 115

GAG

Glu

CAA

Gin

ATA

Ile

AAC

Asn

GTO

Val 195

ATG

Met Gly

CAG

Gin

OTO

Leu

CAG,

Gin

ATO

Ile 180

GAO

Asp

ATO

Ile Giu

CAG

Gin

ATG

Met

GAG

Giu 165

OTA

Leu

GAT

Asp

GOT

Ala Glu

OTT

Leu

AAT

Asn iso

GAA

Giu

AAG

Lys

GTA

Val

CAT

His Leu

GAG

Glu 135

GAG

Giu

AAC

Asn

ACA

Thr

OCA

Pro

CAG,

GAA CCA Giu Pro 120 ACT GCT Thr Ala TOO OTO Ser Leu AGO ATG Ser Met AAA CAA Lys Gin 185 CAG OCA Gin Pro 200 ACT TOT

ATG

Met

OTT

Leu

AAO

Asn

OTT

Leu 170

ACC

Thr

CAA

Gin

COT

AGO

Ser

AAG

Lys

CAC

His 155

ACC

Thr

CA.A

Gin

OCA

Pro

TTO

Phe

AGG

CTO

Leu

CAC

His 14 0

OTO

Leu

AAA

Lys

TGT

Cys

TTT

Phe

OTA

Leu 220

AGG

AAG

Lys 125

ATT

Ile

CAA

Gin

CAG

Gin

GAG

Giu

CAA

Gin 205

AAT

Asn

AAO

Asn 384 432, 480 528 576 624 672 720 Gin Thr Ser Pro CAA GGA GAA GAO CAA AOG Gin Gly Giu Asp Gin Thr 230 GOG ATG Ala Met Arg Arg 235 AAT OTG GAT OTG ACT OTT GAA COO ATT TAO AAT TAO OTT GGO TGT TAO Asn Leu Asp Leu Thr Leu Giu Pro Ile Tyr Asn Tyr Leu Gly Cys Tyr 240 245 250 GOC GOT T GANN Ala Ala 255 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 255 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:iO: Met Gly Arg Gly Arg Val Giu Leu Lys Arg Ile Glu Asn Lys Ile Asn 1 5 1~0 Arg Gin Val Thr Phe Ser Lys Arg Arg Thr Gly Leu Leu Lys Lys Ala 25 Gin Giu Ile Ser Val Leu Cys Asp Ala Giu Val Ser Leu Ile Val Phe 40 Ser His Lys Gly Lys Leu Phe Giu Tyr Ser Ser Glu Ser Cys Met Giu 55 WO 97/46078 PCT/US96/09453 130 Lys Val Leu Glu Arg Tyr Glu Arg Tyr Ser Tyr Ala Glu Arg Gin Leu 70 75 Ile Ala Pro Asp Ser His Val Asn Ala Gin Thr Asn Trp Ser Met Glu 90 Tyr Ser Arg Leu Lys Ala Lys Ile Glu Leu Leu Glu Arg Asn Gin Arg 100 105 110 His Tyr Leu Gly Glu Glu Leu Glu Pro Met Ser Leu Lys Asp Leu Gin 115 120 125 Asn Leu Glu Gin Gin Leu Glu Thr Ala Leu Lys His Ile Arg Ser Arg 130 135 140 Lys Asn Gin Leu Met Asn Glu Ser Leu Asn His Leu Gin Arg Lys Glu 145 150 155 160 Lys Glu Ile Gin Glu Glu Asn Ser Met Leu Thr Lys Gin Ile Lys Glu 165 170 175 Arg Glu Asn Ile Leu Lys Thr Lys Gin Thr Gin Cys Glu Gin Leu Asn 180 185 190 Arg Ser Val Asp Asp Val Pro Gin Pro Gin Pro Phe Gin His Pro His 195 200 205 Leu Tyr Met Ile Ala His Gin Thr Ser Pro Phe Leu Asn Met Gly Gly 210 215 220 Leu Tyr Gin Gly Glu Asp Gin Thr Ala Met Arg Arg Asn Asn Leu Asp 225 230 235 240 Leu Thr Leu Glu Pro Ile Tyr Asn Tyr Leu Gly Cys Tyr Ala Ala 245 250 255 INFORMATION FOR SEQ ID NO:11: SEQUENCE CHARACTERISTICS: LENGTH: 756 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..754 (ix) FEATURE: NAME/KEY: misc_feature LOCATION: 1..756 OTHER INFORMATION: /note= "product Brassica oleracea

CAL."

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: ATG GGA AGG GGT AGG GTT GAA ATG AAG AGG ATA GAG AAC AAG ATC AAC 48 Met Gly Arg Gly Arg Val Glu Met Lys Arg Ile Glu Asn Lys Ile Asn 1 5 10 CGA CAA GTG ACG TTT TCG AAA AGA AGA GCT GGT CTT TTG AAG AAA GCC 96 Arg Gin Val Thr Phe Ser Lys Arg Arg Ala Gly Leu Leu Lys Lys Ala 25 PCT/US96/09453 WO 97/46078 131 CAT GAG ATC His Glu Ile TCG ATC CTT TGT Ser Ile Leu Cys

GAT

Asp 40 GCT GAG GTT TCC CTT ATT GTC TTC Ala Glu Val Ser Leu Ile Val Phe TCC CAT Ser His AAG GGG AAA CTG Lys Gly Lys Leu

TTC

Phe 55 GAG TAC TCG TCT GAA TCT TGC ATG GAG Glu Tyr Ser Ser Glu Ser Cys Met Glu

AAG

Lys GTA CTA GAA CAC Val Leu Glu His

TAC

Tyr GAG AGG TAC TCT Glu Arg Tyr Ser GCC GAG AAA CAG Ala Glu Lys Gin 240 AAA GTT CCA GAC Lys Val Pro Asp

TCT

Ser CAC GTC AAT GCA His Val Asn Ala

CAA

Gin 90 ACG AAC TGG TCA Thr Asn Trp Ser GTG GAA Val Glu TAT AGC AGG Tyr Ser Arg CAT TAT CTG His Tyr Leu 115

CTT

Leu 100 AAG GCT AAG ATT Lys Ala Lys Ile CTT TTG GAG AGA Leu Leu Glu Arg AAC CAA AGG Asn Gin Arg 110 GAG CTA CAG Glu Leu Gin GGC GAA GAT TTA Gly Glu Asp Leu

GAA

Glu 120 TCA ATC AGC ATA Ser Ile Ser Ile AAT CTG Asn Leu 130 GAG CAG CAG CTT Glu Gin Gin Leu ACT TCT CTT AAA Thr Ser Leu Lys

CAT

His 140 ATT CGC TCG AGA Ile Arg Ser Arg 336 384 432 480 528

AAA

Lys 145 AAT CAA CTA ATG Asn Gin Leu Met GAG TCC CTC AAC Glu Ser Leu Asn CTC CAA AGA AAG Leu Gin Arg Lys AAA GAA ATA CTG Lys Glu Ile Leu

GAG

Glu 165 GAA AAC AGC ATG Glu Asn Ser Met

CTT

Leu 170 GCC AAA CAG ATA Ala Lys Gin Ile AGG GAG Arg Glu 175 AGG GAG AGT Arg Glu Ser CGC AGC CAC Arg Ser His 195

ATC

Ile 180 CTA AGG ACA CAT Leu Arg Thr His AAC CAA TCA GAG Asn Gin Ser Glu CAG CAA AAC Gin Gin Asn 190 AAT CCT TAC Asn Pro Tyr CAT GTA GCT CCT His Val Ala Pro

CAG

Gin 200 CCG CAA CCG CAG Pro Gin Pro Gin

TTA

Leu 205 ATG GCA Met Ala 210 TCA TCT CCT TTC Ser Ser Pro Phe

CTA

Leu 215 AAT ATG GGT Asn Met Gly GGC ATG Gly Met 220 TAC CAA GGA GAA Tyr Gin Gly Glu

TAT

Tyr 225 CCA ACG GCG GTG Pro Thr Ala Val

AGG

Arg 230 AGG AAC CGT CTC Arg Asn Arg Leu GAT CTG ACT CTT GAA CCC Asp Leu Thr Leu Glu Pro 235 240 ATT TAC AAC TGC AAC CTT GGT TAC TTT Ile Tyr Asn Cys Asn Leu Gly Tyr Phe 245 GCC GCA T GA Ala Ala 250 INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 251 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein 1, WO 97/46078 WO 9746078PCT/US96/09453 Met 1 Arg His Ser Lys Lys Tyr His Asn Lys 145 Lys ArC- Arc Mel Ty: 22~ 11.

(2 1 (xi) SEQUENCE DESCRIPTION: SEQ ID N Gly Arg Giy Arg Val Glu Met Lys Arg I 5 10 Gin Val Thr Phe Ser Lys Arg Arg Ala G 25 Giu Ile Ser Ile Leu Cys Asp Ala Glu V~ 40 His Lys Gly Lys Leu Phe Giu Tyr Ser S 55 Val Leu Glu His Tyr Glu Arg Tyr Ser 'J 70 Val Pro Asp Ser His Val Asn Ala Gin 90 Ser Arg Leu Lys Aia Lys Ile Glu Leu 100 105 Tyr Leu Gly Giu Asp Leu Giu Ser Ile 115 120 Leu Giu Gin Gin Leu Asp Thr Ser Leu 130 135 Asn Gin Leu Met His Glu Ser Leu Asn 150 Giu Ile Leu Giu Glu Asn Ser Met Leu 165 170 Giu Ser Ile Leu Arg Thr His Gin Asn 180 185 ;Ser His His Val Ala Pro Gin Pro Gin 195 200 =Ala Ser. Ser Pro Phe Leu Asn Met Gly 210 215 r Pro Thr Ala Val Arg Arg Asn. Arg Leu 5 230 e Tyr Asn Cys Asn Leu Gly Tyr Phe Ala 245 250 INFORMATION FOR SEQ ID NO:13: SEQUENCE CHARACTERISTICS: LENGTH: 756 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 1.-451 32 0:12: le Giu Asn Lys Ile A ly Leu Leu Lys Lys A ral. Ser Leu Ile Val P ~er Glu Ser Cys Met C yr Ala Giu Lys Gin I 75 rhr Asn Trp Ser Val Leu Giu Arg Asn Gin 110 Ser Ile Lys Glu Leu 125 Lys His Ile Arg Ser 140 His Leu Gin Arg Lys 155 Ala Lys Gin Ile Arg 175 Gin Ser Glu Gin Gin 190 Pro Gin Leu Asn Pro 205 Giy Met Tyr Gin Gly 220 Asp Leu Thr Leu Giu 235 Ala .sn ~la ~he ;lu 1 eu s0 1,u krg Gln Arg Glu 160 Glu Asn Tyr Glu Pro 240 WO 97/46078 PCT[US96/09453 133 (ix) FEATURE: NAME/KEY: misc feature LOCATION: 1..756 OTHER INFORMATION: /note= "product Brassica oieracea var. botrytis CAL." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: ATG GGA AGG GGT AGG GTT GAA ATG AAG AGG ATA GAG AAC AAG ATC AAC Met Gly Arg Giy Arg Val Giu Met Lys Arg Ile Giu Asn Lys Ile Asn 1 5 10 AGA CAA GTG ACG TTT TCG AAA Arg Gin Val Thr Phe Ser Lys AGA AGA Arg Arg 25 GCT GGT CTT TTG Ala Gly Leu Leu AAG AAA GCC Lys Lys Ala ATT GTC TTC Ile Val Phe CAT GAG ATC His Giu Ile TCG ATT CTT TGT GAT GCT GAG GTT TCC Ser Ile Leu Cys Asp Ala Giu Vai Ser 144 TCC CAT Ser His AAG GGG AAA CTG Lys Gly Lys Leu GAG TAC TCG TCT GAA TCT TGC ATG GAG Glu Tyr Ser Ser Giu Ser Cys Met Glu

AAG

Lys GTA CTA GAA CGC Val Leu Giu Arg

TAC

Tyr 70 GAG AGG TAC TCT Glu Arg Tyr Ser

TAC

Tyr 75 GCC GAG AAA CAG Ala Giu Lys Gin

CTA

Leu 240 AAA GCT CCA GAC Lys Ala Pro Asp

TCT

Ser CAC GTC AAT OCA His Val Asn Ala ACG AAC TGG TCA Thr Asn Trp Ser ATG GAA Met Giu TAT AGC AGG Tyr Ser Arg CAT TAT CTG His Tyr Leu 115

CTT

Leu 100 AAG GCT AAG ATT Lys Ala Lys Ile CTT TGG GAG AGG Leu Trp Giu Arg AAC CAA AGG Asn Gin Arg 110 GAG CTA CAG Glu Leu Gin 336 GGA GAA GAT TTA GAA TCA ATC AGC ATA Gly Glu Asp Leu Glu Ser Ile Ser Ile

AAG

Lys 125 AAT CTG Asn Leu 130 GAG CAG CAG CTT Glu Gin Gin Leu ACT TCT CTT Thr Ser Leu AAA CAT Lys His 140 ATT CGC TCC AGA Ile Arg Ser Arg

AAA

Lys 145 AAT CAA CTA ATG Asn Gin Leu Met CAC T AGTCCCTCAA CCACCTCCAA AGAAAGGAGA His 150 AAGAAATACT GGAGGAAAAC AGCATGCTTG CCAAACAGAT AAAGGAGAGG GAGAGTATCC TAAGGACACA TCAAAACCAA TCAGAGCAGC AAAACCGCAG CCACCATGTA

GCTCCTCAGC

CGCAACCGCA GTTAAATCCT TACATGGCAT CATCTCCTTT CCTAAATATG GGTGGCATGT ACCAAGGAGA ATATCCAACG GCGGTGAGGA GGAACCGTCT CGATCTGACT CTTGAACCCA 481 541 601 661 721 TTTACAACTG CAACCTTGGT TACTTTGCCG CATGA 756 INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 150 amino acids TYPE: amino acid TOPOLOGY: linear WO 97/46078 PCT/US96/09453 134 (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: Met Gly Arg Gly Arg Val Glu Met Lys Arg Ile Glu Asn Lys Ile Asn 1 5 10 Arg Gin Val Thr Phe Ser Lys Arg Arg Ala Gly Leu Leu Lys Lys Ala 25 His Glu Ile Ser Ile Leu Cys Asp Ala Glu Val Ser Leu Ile Val Phe 40 Ser His Lys Gly Lys Leu Phe Glu Tyr Ser Ser Glu Ser Cys Met Glu 55 Lys Val Leu Glu Arg Tyr Glu Arg Tyr Ser Tyr Ala Glu Lys Gin Leu 70 75 Lys Ala Pro Asp Ser His Val Asn Ala Gin Thr Asn Trp Ser Met Glu 90 Tyr Ser Arg Leu Lys Ala Lys Ile Glu Leu Trp Glu Arg Asn Gin Arg 100 105 110 His Tyr Leu Gly Glu Asp Leu Glu Ser Ile Ser Ile Lys Glu Leu Gin 115 120 125 Asn Leu Glu Gin Gin Leu Asp Thr Ser Leu Lys His Ile Arg Ser Arg 130 135 140 Lys Asn Gin Leu Met His 145 150 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 1500 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 72..1343 (ix) FEATURE: NAME/KEY: misc_feature LOCATION: 1..1500 OTHER INFORMATION: /note= "product Arabidopsis thaliana LEAFY (LFY)." (xi) SEQUENCE DESCRIPTION: SEQ ID AAAGCAATCT GCTCAAAAGA GTAAAGAAAG AGAGAAAAAG AGAGTGATAG AGAGAGAGAG AAAAATAGAT T ATG GAT CCT GAA GGT TTC ACG AGT GGC TTA TTC CGG TGG 110 Met Asp Pro Glu Gly Phe Thr Ser Gly Leu Phe Arg Trp 1 5 AAC CCA ACG AGA GCA TTG GTT CAA GCA CCA CCT CCG GTT CCA CCT CCG 158 Asn Pro Thr Arg Ala Leu Val Gin Ala Pro Pro Pro Val Pro Pro Pro 20 WO 97/46078 PCT/US96/09453 135

CTG

Leu CAG CAA CAG CCG Gin Gin Gin Pro GTG ACA Val Thr CCG CAG ACG Pro Gin Thr GCT TTT GGG ATG Ala Phe Gly Met CTT GGT GGT TTA Leu Gly Gly Leu GGA CTA TTC GGT Gly Leu Phe Gly

CCA

Pro 55 TAC GGT ATA CGT Tyr Gly Ile Arg TTC TAC Phe Tyr ACG GCG GCG Thr Ala Ala GGT ATG AAG Gly Met Lys ATA GCG GAG TTA Ile Ala Giu Leu

GGT

Gly 70 TTT ACG GCG AGC Phe Thr Ala Ser ACG CTT GTG Thr Leu Val CTC TCT CAT Leu Ser His GAC GAG GAG CTT Asp Giu Glu Leu GAG ATG ATG AAT Glu Met Met Asn ATC TTT Ile Phe CGT TGG GAG CTT Arg Trp Glu Leu GTT GGT GAA CGG Val Gly Giu Arg

TAC

Tyr 105 GGT ATC AAA GCT Gly Ile Lys Ala

GCC

Ala 110 GTT AGA GCT GAA Val Arg Ala Glu AGA CGA TTG CAA Arg Arg Leu Gin

GAA

Glu 120 GAG GAG GAA GAG Glu Glu Glu Glu

GAA

Glu 125 TCT TCT AGA CGC Ser Ser Arg Arg CAT TTG CTA CTC His Leu Leu Leu

TCC

Ser 135 GCC GCT GGT GAT Ala Ala Gly Asp TCC GGT Ser Gly 140 ACT CAT CAC Thr His His TTA TCT GAG Leu Ser Glu 160

GCT

Ala 145 CTT GAT GCT CTC Leu Asp Ala Leu

TCC

Ser 150 CAA GAA GAT GAT Gin Giu Asp Asp TGG ACA GGG Trp Thr Gly 155 GCG GCG GGG Ala Ala Gly 542 590 GAA CCG GTG CAG Glu Pro Val Gin

CAA

Gin 165 CAA GAC CAG ACT Gin Asp Gin Thr

GAT

Asp 170 AAT AAC GGC GGA GGA GGA AGT GGT TAC TGG GAC OCA GGT CAA GGA AAG Asn Asn Gly Gly Gly Gly Ser Gly Tyr Trp Asp Ala Gly Gin Gly Lys 175 180 185 ATG AAG AAG CAA CAG CAG CAG AGA CGG AGA AAG AAA CCA ATG CTG ACG Met Lys Lys Gin Gin Gin Gin Arg Arg Arg Lys Lys Pro Met Leu Thr 190 195 200 205 TCA GTG GAA ACC Ser Val Glu Thr

GAC

Asp 210 GAA GAC GTC AAC Glu Asp Val Asn

GAA

Glu 215 GGT GAG GAT GAC Gly Glu Asp Asp GAC GGG 734 Asp Gly 220 ATG GAT AAC Met Asp Asn GAG CAT CCG Glu His Pro 240

GGC

Gly 225 AAC GGA GGT AGT Asn Gly Gly Ser

GGT

Gly 230 TTG GGG ACA GAG Leu Gly Thr Glu AGA CAG AGG Arg Gin Arg 235 CGT GGC AAA Arg Gly Lys TTT ATC GTA ACG Phe Ile Val Thr

GAG

Glu 245 CCT GGG GAA GTG Pro Gly Glu Val

GCA

Ala 250 AAG AAC GGC TTA GAT TAT CTG TTC CAC TTG TAC GAA CAA TGC CGT GAG Lys Asn Gly Leu Asp Tyr Leu Phe His Leu Tyr Glu Gin Cys Arg Glu 255 260 265 TTC CTT CTT CAG Phe Leu Leu Gin 270 CCC ACC AAG GTG Pro Thr Lys Val

CAG

Gin 275 ACA ATT GCT AAA Thr Ile Ala Lys

GAC

Asp 280 CGT GGC GAA AAA Arg Gly Giu Lys

TGC

Cys 285 ACG AAC CAA GTA TTC Thr Asn Gin Val Phe 290 AGG TAC GCG AAG AAA Arg Tyr Ala Lys Lys 295 TCA GGA Ser Gly 300 PCT/US96/09453 c WO 97/46078 136 GCG AGT TAC ATA AAC AAG CCT AAA ATG CGA CAC TAC GT Ala Ser Tyr Ile Asn Lys Pro Lys Met Arg His Tyr Va 305 310 GCT CTC CAC TGC CTA GAC GAA GAA GCT TCA AAT GCT C9 Ala Leu His Cys Leu Asp Glu Glu Ala Ser Asn Ala Lf 320 325 3; TTT AAA GAA CGC GGT GAG AAC GTT GGC TCA TGG CGT C Phe Lys Glu Arg Gly Glu Asn Val Gly Ser Trp Arg G 335 340 345 AAG CCA CTT GTG AAC ATC GCT TGT CGT CAT GGC TGG GJ Lys Pro Leu Val Asn Ile Ala Cys Arg His Gly Trp Al 350 355 360 GTC TTT AAC GCT CAT CCT CGT CTC TCT ATT TGG TAT G Val Phe Asn Ala His Pro Arg Leu Ser Ile Trp Tyr V 370 375 CTG CGT CAG CTT TGC CAT TTG GAG CGG AAC AAT GCG G Leu Arg Gin Leu Cys His Leu Glu Arg Asn Asn Ala V 385 390 GCG GCT TTA GTT GGC GGT ATT AGC TGT ACC GGA TCG T Ala Ala Leu Val Gly Gly Ile Ser Cys Thr Gly Ser S 400 405 4 CGT GGT GGA TGC GGC GGC GAC GAC TTG CGT TTC TAGTT Arg Gly Gly Cys Gly Gly Asp Asp Leu Arg Phe 415 420 TGGTTTGTTT AGTCGTTATC CTAATTAACT ATTAGTCTTT AATT TTTATTTTTC TTTTTTTGTC AAAACCTTTA ATTTGTTATG GCTAI GTTTTCTTAA TGCGTTA INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 424 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: Met Asp Pro Glu Gly Phe Thr Ser Gly Leu Phe Arg 1 5 10 Arg Ala Leu Val Gin Ala Pro Pro Pro Val Pro Pro 25 Gin Pro Val Thr Pro Gin Thr Ala Ala Phe Gly Met 40 Leu Glu Gly Leu Phe Gly Pro Tyr Gly Ile Arg Phe 55 Lys Ile Ala Glu Leu Gly Phe Thr Ala Ser Thr Leu 70 75 Asp Glu Glu Leu Glu Glu Met Met Asn Ser Leu Ser 90 T CAC il His 315 :C AGA eu Arg AG GCT in Ala AT ATA sp Ile TT CCA al Pro TT GCT al Ala 395 CG ACG er Thr

TGGTT

'AGTCT

ATTTGT

TGT TAC Cys Tyr AGA GCG Arg Ala TGT TAC Cys Tyr GAC GCC Asp Ala 365 ACA AAG Thr Lys 380 GCG GCT Ala Ala TCT GGA Ser Gly

TGGGTAGTTG

TCTTGGCTAA

TATACACGCA

1022 1070 1118 1166 1214 1262 1310 1363 1423 1483 1500 Trp Pro Arg Tyr Val His Asn Leu Leu Thr Gly Ile Pro Thr Gin Gin Gly Gly Ala Ala Met Lys Phe Arg PCTUS96/09453 WO 97/46078 Trp G: Ala G Arg A 1 Ala L 145 Glu P Gly G Gin G Thr Gly 225 Phe Leu Gin Val Ile 305 Cys Arg Val Ala Leu 385 Val Cys Lu lu rg 30 eu ro Ily Iln sp ~sn Ile ssp Val Thr 290 Asr Le.

Gi' AsI Hi 37 Cy Gi Gi Leu Leu V 100 Arg Arg A~ 115 His Leu L Asp Ala L Val Gin G 1 Gly Ser G 180 Gin Gin A 195 Glu Asp V Gly Gly S Val Thr C Tyr Leu I 260 Gin Thr 275 Asn Gin Lys Pro i Asp Glu Giu Asn 340 n lie Ala 355 s Pro Arg 0 s His Leu y Gly Ile y Giy Asp 420 in rg eu ln 65 ly rg 'al ;er lu ?he lie al Ly Gl 32 Va Cy Le Gi Se As Gly Giu Al Leu Gin G1 Leu Ser A: 135 Ser Gin G: 150 Gin Asp G Tyr Trp A Arg Arg L 2 Asn Giu G 215 Gly Leu G 230 Pro Giy C His Leu J Ala Lys Phe Arg 295 3 Met Arg 310 a Aia Ser 5 1 Giy Ser s Arg His u Ser Ile 375 u Arg Asn 390 r Cys Thr '5 ;p Leu Arg Lu 20 la lu in sp ,ys 00 ly ;ly ;lu [yr ksp 28C Tyi Hi As Tr Gi 36 Tr As Gi P1.

Tyr G 105 Glu G: Ala G Asp A Thr A 1 Ala G 185 Lys P Glu P Thr C Val 2 Glu 265 Arg Ala 3 Tyr a Ala p Arg 345 y Trp 0 p Tyr n Ala v Ser Ly Lu ly sp sp 70 ly ro sp 311 Z 5 31' Ly Va Le 33

GI

As Va

V

Si 4: 137 Ile L) Glu G: Asp S Trp T 155 Ala A Gln G Met L Asp A 2 Arg G 235 Arg C i Cys I y Glu s Lys 1 His 315 u Arg 0 n Ala ;p Ile 1 Pro il Ala 395 er Thr

LO

'5 Lu er hr la ly eu sp Iln fly krg ys Sei CyE Ar Cy As Th 38 Al Se Ala Ala Vi 110 Glu Ser S 125 Gly Thr H Gly Leu S Gly Asn A 1 Lys Met L 190 Thr Ser Ni 205 Gly Met I Arg Giu I Lys Lys Glu Phe 270 Cys Pro 285 Gly Ala 3 Tyr Ala 3 Ala Phe s Tyr Lys 350 p Ala Val 365 r Lys Leu 0 a Aia Ala r Giy Arg al Arg er Arg is His er Glu 160 sn Gly lys Lys ral Glu ~sp Asn Us Pro 240 ksn Gly 255 Ueu Leu Thr Lys Ser Tyr Leu His 320 Lys Glu 335 Pro Leu Phe Asn Arg Gin Ala Leu 400 Gly Gly 415 ee PCT/US96/09453 IIWO 97/46078 138 INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 1656 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..1651 (ix) FEATURE: NAME/KEY: misc feature LOCATION: 1..1656 OTHER INFORMATION: /note= "domain ligand binding domain." ecdysone receptor (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: CGG CCG GAA TGC GTC GTC CCG GAG AAC CAA TGT GCG ATG AAG CGG Arg Pro Glu Cys Val Val Pro Glu Asn Gin Cys Ala Met Lys Arg 5 10

ATG

Met 1 CGC GAA AAG AAG GCC CAG AAG GAG AAG Arg Glu Lys Lys Ala Gin Lys Glu Lys 25 GAC AAA ATG ACC Asp Lys Met Thr ACT TCG CCG Thr Ser Pro GGC GGC CAA Gly Gly Gin AGC TCT CAG Ser Ser Gin CAT GGC GGC AAT His Gly Gly Asn

GGC

Gly AGC TTG GCC TCT Ser Leu Ala Ser GAC TTT Asp Phe GTT AAG AAG GAG ATT CTT GAC CTT ATG Val Lys Lys Glu Ile Leu Asp Leu Met 55

ACA

Thr TGC GAG CCG CCC Cys Glu Pro Pro

CAG

Gin CAT GCC ACT ATT His Ala Thr Ile CTA CTA CCT GAT Leu Leu Pro Asp

GAA

Glu ATA TTG GCC AAG Ile Leu Ala Lys 240 CAA GCG CGC AAT Gin Ala Arg Asn

ATA

Ile CCT TCC TTA ACG Pro Ser Leu Thr

TAC

Tyr 90 AAT CAG TTG GCC Asn Gin Leu Ala GTT ATA Val Ile TAC AAG TTA Tyr Lys Leu GAT CTC AGG Asp Leu Arg 115

ATT

Ile 100 TGG TAC CAG GAT Trp Tyr Gin Asp

GGC

Gly 105 TAT GAG CAG CCA TCT GAA GAG Tyr Glu Gin Pro Ser Glu Glu 110 CGT ATA ATG AGT Arg Ile Met Ser CCC GAT GAG AAC Pro Asp Glu Asn AGC CAA ACG Ser Gin Thr GAC GTC Asp Val 130 AGC TTT CGG CAT Ser Phe Arg His ATA ACC Ile Thr 135 GAG ATA ACC ATA CTC ACG GTC CAG Glu Ile Thr Ile Leu Thr Val Gin 140 TTG ATT Leu Ile 145 GTT GAG TTT Val Glu Phe

GCT

Ala 150 AAA GGT CTA CCA Lys Gly Leu Pro

GCG

Ala 155 TTT ACA AAG ATA Phe Thr Lys Ile CAG GAG GAC CAG ATC ACG TTA CTA AAG GCC Gin Glu Asp Gin Ile Thr Leu Leu Lys Ala 165 170 TGC TCG TCG GAG Cys Ser Ser Glu GTG ATG 528 Val Met 175 PCTIUS96/09453 WO 97/46078 139 ATG CTG OGT Met Leu Arg TTO GCG AAT Phe Ala Asn 195 GOA CGA CGC TAT Ala Arg Arg Tyr

GAC

Asp 185 CAC AGO TOG GAO His Ser Ser Asp TCA ATA TTO Ser Ile Pile 190 ATG GCC GGA Met Ala Gly AAT AGA TCA TAT Asn Arg Ser Tyr

AOG

Thr 200 CGG GAT TOT TAO Arg Asp Ser Tyr

AAA

Lys 205 ATG GOT Met Ala 210 GAT AAC ATT GAA Asp Asn Ile Glu

GAO

Asp 215 OTG OTG CAT TTO Leu Leu His Pile

TGO

Cys 220 OGO CAA ATG TTC Arg Gin Met Pile ATG AAG GTG GAO Met Lys Val Asp

AAC

Asfl 230 GTO GAA TAO GCG Val Giu Tyr Ala OTO ACT GOO ATT Leu Thr Ala Ile ATO TTO TOG GAO Ile Pile Ser Asp

OGG

Arg 245 OOG GGO OTG GAG Pro Gly Leu Giu

AAG,

Lys 250 GOO CAA OTA GTC Ala Gin Leu Val GAA GOG Glu Ala 255 ATO CAG AGO Ilie Gin Ser CAC TGO GGO His Cys Gly 275

TAO

Tyr 260 TAO ATO GAO ACG Tyr Ilie Asp Thr

OTA

Leu 265 OGO ATT TAT ATA Arg Ile Tyr Ile OTO AAO OGO Leu Asn Arg 270 CTG OTO TOG Leu Leu Ser GAC TOA ATG AGO Asp Ser Met Ser

CTO

Leu 280 GTC TTO TAO GOA Val Phe Tyr Ala

AAG

Lys 285 ATO OTO Ile Leu 290 ACC GAG OTG OGT Thr Glu Leu Arg OTG GGC AAO CAG Leu Gly Asn Gin

AAO

Asn 300 GOC GAG ATG TGT Ala Glu Met Cys

TTO

Pile 305 TOA OTA AAG OTO Ser Leu Lys Leu AAO OGO AAA CTG Asn Arg Lys Leu AAG TTC OTO GAG Lys Pile Leu Glu

GAG

Giu 320 ATO TGG GAO GTT Ile Trp Asp Val GOC ATO COG OCA Ala Ilie Pro Pro

TOG

Ser 330 GTO CAG TOG CAC Val Gin Ser His OTT CAG Leu Gin 335 ATT ACC CAG Ile Thr Gin GOA TOG GTT Ala Ser Val 355 GAG AAC GAG OGT Giu Asn Giu Arg

OTO

Leu 345 GAG OGG GOT GAG Giu Arg Ala Giu CGT ATG CGG Arg Met Arg 350 GAO TOT GC Asp Ser Ala 816 864 912 960 1008 1056 1104 1152 1200 1248 1296 1344 GGG GGO GOC ATT Gly Gly Ala Ile GOC GGO ATT GAT Ala Gly Ile Asp TOO ACT Ser Thr 370 TOG GOG GCG GCA Ser Ala Ala Ala

GC

Ala 375 GOG GOC CAG OAT Ala Ala Gin His COT CAG COT CAG Pro Gin Pro Gin CAG COO CAA 000 Gin Pro Gin Pro

TOO

Ser 390 TOO CTG ACC CAG Ser Leu Thr Gin

AAC

Asn 395 GAT TOO CAG CAC Asp Ser Gin His

CAG

Gin 400 ACA CAG COG CAG Thr Gin Pro Gin

OTA

Leu 405 CAA COT CAG OTA Gin Pro Gin Leu

OCA

Pro 410 OCT CAG OTG CAA Pro Gin Leu Gin GGT CAA Gly Gin 415 OTG CAA COO Leu Gin Pro

CAG

Gin 420 OTO CAA OCA CAG Leu Gin Pro Gin

OTT

Leu 425 CAG AOG CAA OTO CAG OCA CAG Gin Thr Gin Leu Gin Pro Gin 430 ATT CAA OCA CAG OCA CAG OTO Ilie Gin Pro Gin Pro Gin Leu CCC GTO TOO GOT 000 GTG COO GC Pro Val Ser Ala Pro Val Pro Ala 445 PCTIEJS96/09453 WO 97/46078 14 TCC G Ser VJ 4

TAC

Tyr P~ 465

AGC

Ser

CCG

Pro

AGC

Ser

CTG

Leu

GAG

Glu 545 (2) Met Arg Ser Asp Gin Gir Ty Asi As TA ACC GCA CCT GGT TCC TTG TCC GLLG G1 'al Thr Ala Pro Gly Ser Leu Ser Ala Va 50 455 TG GGC GGA AGT GCG GCC ATA GGA CCC AT let Gly Gly Ser Ala Ala Ile Gly Pro Il 470 47 GTATC ACG GCT GCC GTT ACC GCT AGC TC 3er Ile Thr Ala Ala Val Thr Ala Ser Se 485 490 %TG GOC AAC GGA GTT GGA GTC GGT GTT GC 4let Gly Asn Gly Val Gly Val. Gly Val Gl 500 505 P.TG TAT GCG AAC GCC CAG ACG GCG ATG G( Met Tyr Ala Asn Ala Gin Thr Ala Met A 515 520 CAT TCG CAC CAA GAG CAG CTT ATC GGG G( His Ser His Gin Glu Gin Leu Ile Gly G 530 535 CAC TCG ACG ACT GCA T AGCAG His Ser Thr Thr Ala 550 INFORMATION FOR SEQ ID NO:l8: SEQUENCE CHARACTERISTICS: LENGTH: 550 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID I Arg Pro Giu Cys Val. Val. Pro Glu Asn 10 Glu Lys Lys Ala Gin Lys Giu Lys Asp 25 Ser Gin His Gly Gly Asn Gly Ser Leu 40 Phe Val. Lys Lys Glu Ile Leu Asp Leu 55 His Ala Thr Ile Pro Leu Leu Pro Asp 70 1Ala Arg Asn Ile Pro Ser Leu Thr Tyr 90 Lys Leu Ile Trp Tyr Gin Asp Gly Tyr 100 105 SLeu Arg Arg Ile Met Ser Gin Pro Asp 115 '120 p Val. Ser Phe Arg His Ile Thr Glu Ile 130 135 0

C.

e 5

C

ly

DGT

Ser 460

ACG

Thr

ACC

Thr

GTG

Val

TTG

Leu

GTG

Val.

540

ACG

Thr

CCG

Pro

ACA

Thr

GGC

Giy

ATG

Met 525

GCG

Ala AGC AGC Ser Ser GCA ACC Ala Thr TCA GCG Ser Ala 495 GGC AAC Gly Asn 510 GGT GTA Gly Va).

GTT AAG Val Lys

GAA

Glu

ACC

Thr 480

GTA

Val.

GTC

Val.

GCC

Ala

TCG

Ser 1392 1440 1488 1536 1584 1632 16S6 0O:18: in Cys Ala Met Lys Met Thr Thr Ala Ser Gly Gly Met Thr Cys G).u Giu Ile L~eU Ala 75 Asn Gin Leu Ala Giu Gin Pro Ser Giu Asn Giu Ser 125 Thr Ile Leu Thr 140 Lys is Ser Gly Pro Lys Val.

Giu Gin Val Arg Pro Gin Pro Cys Ile *Giu Thr -Gin PCT/US96/09453 WO 97/46078 Leu Ile Val G1 145 Gin Glu Asp G1 Met Leu Arg Me 1I Phe Ala Asn AE 195 Met Ala Asp As 210 Ser Met Lys V, 225 Ile Phe Ser A Ile Gin Ser T 2 His Cys Gly A 275 Ile Leu Thr G 290 Phe Ser Leu I 305 Ile Trp Asp t Ile Thr Gin Ala Ser Val 355 Ser Thr Ser 370 Pro Gin Pro 385 Thr Gin Pro Leu Gin Pro Ile Gin Pro 435 Ser Val Thr 450 Tyr Met Gly 465 Ser Ser Ile u .n it )0 sn sn al sp yr 60 .sp lu 311 .ys Val Gli 34 31 Al G1 G1 G1 42 G1

A:

G:

T:

Phe Ala Ly 150 Ile Thr Le 165 Ala Arg Ar Arg Ser Ty Ile Glu AE 2] Asp Asn Vc 230 Arg Pro G: 245 Tyr Ile A Ser Met S Leu Arg T 2 SLeu Lys A 310 L His Ala I 325 u Glu Asn C 0 y Gly Ala a Ala Ala n Pro Ser 390 .n Leu Gin 405 .n Leu Gin !0 .n Pro Gin La Pro Gly ly Ser Ala 470 hr Ala Ala 485 s G1 u Le g Ty 'r Tk 2( sp L 15 al G: Ly L sp T er L 2 hr L 95 sn le I Glu lie Ala 375 Ser Pro Pro Leu Ser 455 Ala Val y u r hr 00 Lu lu eu hr ieu 8C eui ir Ari Th 36 Al Le G1 G1 Li 4

L

I

T

Leu Pr Lys Al 17 Asp Hi 185 Arg As Leu Hi Tyr A] Glu L' 2! Leu A: 265 Val P SGly A g Lys L o Pro S 3 g Leu C 345 r Ala C 0 a Ala !u Thr .n Leu In Leu 425 eu Pro 40 eu Ser le Gly hr Ala o a 0 Ls la' 50 rg he s iel ;e 3 3l 31 G1 G1

P]

4:

G

V

A

P

4 141 Ala Ph 155 Cys Se Ser Se Ser Ty Phe Cy 22 Leu LE 235 SAla G SIle T e Tyr A: n Gin A 3 u Pro L 315 r Val G 0 u Arg A .y Ile I n His Ln Asn 395 ro Pro 10 In Thr al Ser .la Val >ro Ile 475 er Ser 90 e r r r 's 0 eu in yr la sn 00 ys Ilr As 38 As G1 G1 A1

SE

4T

T

Thr Lys Il Ser Glu Va 17 Asp Ser Ii 190 Lys Met Al 205 Arg Gin Me Thr Ala I] Leu Val G: 2! Ile Leu Ai 270 Lys Leu L 285 SAla Glu M Phe Leu G SSer His L 3 a Glu Arg I 350 p Cys Asp 365 n Pro Gin 0 p Ser Gn n Leu Gin .n Leu Gin 430 a Pro Val 445 er Thr Ser hr Pro Ala hr Thr Ser e Pr 16 1 Me e Ph .a Gl it P1 Le V< 2- Lu A sn A: eu S et C lu G 3 eu G let I ser Pro His Gly 415 Pro Pro Ser Thr Ala 495 o 0 t Le *y he al la rg er ys lu l1n Arg 3la Gln Gin 400 Gin Gin Ala Glu Thr 480 Val PCT/US96/09453 WO 97/46078

P

s

L

C

142 ro Met Gly Asn Gly Val Gly Val Gly Val Gly Val Gly Gly Asn Val 500 505 510 er Met Tyr Ala Asn Ala Gin Thr Ala Met Ala Leu Met Gly Val Ala 515 520 525 eu His Ser His Gin Glu Gin Leu Ile Gly Gly Val Ala Val Lys Ser 530 535 540 lu His Ser Thr Thr Ala 545 550 INFORMATION FOR SEQ ID NO:19: SEQUENCE

CHARACTERISTICS:

LENGTH: 855 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..853 (ix) FEATURE: NAME/KEY: misc feature LOCATION: 1..855 OTHER INFORMATION: /note= "domain glucocorticoid receptor ligand binding domain." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: ACA AAG AAA AAA ATC AAA GGG ATT CAG CAA GCC ACT GCA GGA GTC TCA Thr Lys Lys Lys Ile Lys Gly Ile Gin Gin Ala Thr Ala Gly Val Ser 1 5 10 CAA GAC ACT TCG GAA AAT CCT AAC AAA ACA ATA GTT CCT GCA GCA TTA Gin Asp Thr Ser Glu Asn Pro Asn Lys Thr Ile Val Pro Ala Ala Leu 25 CCA CAG CTC ACC CCT ACC TTG GTG TCA CTG CTG GAG GTG ATT GAA CCC Pro Gin Leu Thr Pro Thr Leu Val Ser Leu Leu Glu Val Ile Glu Pro 40 GAG GTG TTG TAT GCA GGA TAT GAT AGC TCT GTT CCA GAT TCA GCA TGG Glu Val Leu Tyr Ala Gly Tyr Asp Ser Ser Val Pro Asp Ser Ala Trp 55 AGA ATT ATG ACC ACA CTC AAC ATG TTA GGT GGG CGT CAA GTG ATT GCA Arg Ile Met Thr Thr Leu Asn Met Leu Gly Gly Arg Gin Val Ile Ala 70 75 GCA GTG AAA TGG GCA AAG GCG ATA CTA GGC TTG AGA AAC TTA CAC CTC Ala Val Lys Trp Ala Lys Ala Ile Leu Gly Leu Arg Asn Leu His Leu 90 GAT GAC CAA ATG ACC CTG CTA CAG TAC TCA TGG ATG TTT CTC ATG GCP Asp Asp Gin Met Thr Leu Leu Gin Tyr Ser Trp Met Phe Leu Met Ala 100 105 110 TTT GCC TTG GGT TGG AGA TCA TAC AGA CAA TCA AGC GGA AAC CTG CTC Phe Ala Leu Gly Trp Arg Ser Tyr Arg Gin Ser Ser Gly Asn Leu Let 115 120 125 1 48 96 144 192 240 288 336 384 WO 97/46078 PCTIUS96/09453 143 TGC TTT GCT CCT GAT CTG ATT ATT AAT GAG CAG AGA ATG TCT CTA CCC 432 Cys Phe Ala Pro Asp Leu Ile Ile Asn Glu Gin Arg Met Ser Leu Pro 130 135 140 TGC ATG TAT GAC CAA TGT AAA CAC ATG CTG TTT GTC TCC TCT GAA TTA 480 Cys Met Tyr Asp Gin Cys Lys His Met Leu Phe Val Ser Ser Glu Leu 145 150 155 160 CAA AGA TTG CAG GTA TCC TAT GAA GAG TAT CTC TGT ATG AAA ACC TTA 528 Gin Arg Leu Gin Val Ser Tyr Glu Glu Tyr Leu Cys Met Lys Thr Leu 165 170 175 CTG CTT CTC TCC TCA GTT GCT AAG GAA GGT CTG AAG AGC CAA GAG TTA 576 Leu Leu Leu Ser Ser Val Ala Lys Glu Gly Leu Lys Ser Gin Glu Leu 180 185 190 TTT GAT GAG ATT CGA ATG ACT TAT ATC AAA GAG CTA GGA AAA GCC ATC 624 Phe Asp Glu Ile Arg Met Thr Tyr Ile Lys Glu Leu Gly Lys Ala Ile 195 200 205 GTC AAA AGG GAA GGG AAC TCC AGT CAG AAC TGG CAA CGG TTT TAC CAA 672 Val Lys Arg Glu Gly Asn Ser Ser Gin Asn Trp Gin Arg Phe Tyr Gin 210 215 220 CTG ACA AAG CTT CTG GAC TCC ATG CAT GAG GTG GTT GAG AAT CTC CTT 720 Leu Thr Lys Leu Leu Asp Ser Met His Glu Val Val Glu Asn Leu Leu 225 230 235 240 ACC TAC TGC TTC CAG ACA TTT TTG GAT AAG ACC ATG AGT ATT GAA TTC 768 Thr Tyr Cys Phe Gin Thr Phe Leu Asp Lys Thr Met Ser Ile Glu Phe 245 250 255 CCA GAG ATG TTA GCT GAA ATC ATC ACT AAT CAG ATA CCA AAA TAT TCA 816 Pro Glu Met Leu Ala Glu Ile Ile Thr Asn Gin Ile Pro Lys Tyr Ser 260 265 270 AAT GGA AAT ATC AAA AAG CTT CTG TTT CAT CAA AAA T GA 855 Asn Gly Asn Ile Lys Lys Leu Leu Phe His Gin Lys 275 280 INFORMATION FOR SEQ ID SEQUENCE

CHARACTERISTICS:

LENGTH: 284 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Thr Lys Lys Lys Ile Lys Gly Ile Gin Gin Ala Thr Ala Gly Val Ser 1 5 10 Gin Asp Thr Ser Glu Asn Pro Asn Lys Thr Ile Val Pro Ala Ala Leu 25 Pro Gin Leu Thr Pro Thr Leu Val Ser Leu Leu Glu Val Ile Glu Pro 40 Glu Val Leu Tyr Ala Gly Tyr Asp Ser Ser Val Pro Asp Ser Ala Trp 55 Arg Ile Met Thr Thr Leu Asn Met Leu Gly Gly Arg Gin Val Ile Ala 70 75 WO 97/46078 PCT/US96/09453 144 Ala Val Lys Trp Ala Lys Ala Ile Leu Gly Leu Arg Asn Leu His Leu 90 Asp Asp Gln Met Thr Leu Leu Gin Tyr Ser Trp Met Phe Leu Met Ala 100 105 110 Phe Ala Leu Gly Trp Arg Ser Tyr Arg Gin Ser Ser Gly Asn Leu Leu 115 120 125 Cys Phe Ala Pro Asp Leu Ile Ile Asn Glu Gin Arg Met Ser Leu Pro 130 135 140 Cys Met Tyr Asp Gin Cys Lys His Met Leu Phe Val Ser Ser Glu Leu 145 150 155 160 Gin Arg Leu Gin Val Ser Tyr Glu Glu Tyr Leu Cys Met Lys Thr Leu 165 170 175 Leu Leu Leu Ser Ser Val Ala Lys Glu Gly Leu Lys Ser Gin Glu Leu 180 185 190 Phe Asp Glu Ile Arg Met Thr Tyr Ile Lys Glu Leu Gly Lys Ala Ile 195 200 205 Val Lys Arg Glu Gly Asn Ser Ser Gin Asn Trp Gin Arg Phe Tyr Gin 210 215 220 Leu Thr Lys Leu Leu Asp Ser Met His Glu Val Val Glu Asn Leu Leu 225 230 235 240 Thr Tyr Cys Phe Gin Thr Phe Leu Asp Lys Thr Met Ser Ile Glu Phe 245 250 255 Pro Glu Met Leu Ala Glu Ile Ile Thr Asn Gin Ile Pro Lys Tyr Ser 260 265 270 Asn Gly Asn Ile Lys Lys Leu Leu Phe His Gin Lys 275 280 INFORMATION FOR SEQ ID NO:21: SEQUENCE CHARACTERISTICS: LENGTH: 50 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ix) FEATURE: NAME/KEY: misc_feature LOCATION: 1..50 OTHER INFORMATION: /note= "element copper inducible regulatory element (ACE1 binding site)." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: AGCTTAGCGA TGCGTCTTTT CCGCTGAACC GTTCCAGCAA AAAAGACTAG INFORMATION FOR SEQ ID NO:22: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear WO 97/46078 PCT/US96/09453 145 (ix) FEATURE: NAME/KEY: misc feature LOCATION: 1..19 OTHER INFORMATION: /note= "element tet operator." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: ACTCTATCAG TGATAGAGT 19 INFORMATION FOR SEQ ID NO:23: SEQUENCE

CHARACTERISTICS:

LENGTH: 29 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ix) FEATURE: NAME/KEY: misc_feature LOCATION: 1..29 OTHER INFORMATION: /note= "element ecdysone response element." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: GATCCGACAA GGGTTCAATG CACTTGTCA 2! INFORMATION FOR SEQ ID NO:24: SEQUENCE

CHARACTERISTICS:

LENGTH: 371 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ix) FEATURE: NAME/KEY: misc_feature LOCATION: 1..371 OTHER INFORMATION: /note= "element heat shock inducible regulatory element (HSP81-1 promoter)." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: GTGGAGTCTC GAAACGAAAA GAACTTTCTG GAATTCGTTT GCTCACAAAG

CTAAAAACGG

TTGATTTCAT CGAAATACGG CGTCGTTTTC AAAGAACAAT CCAGAAATCA

CTGGTTTTCC

TTTATTTCAA AAGAAGAGAC TAGAACTTTA TTTCTCCTCT ATAAAATCAC

TTTGTTTTTC

CCTCTCTTCT TCATAAATCA ACAAAACAAT CACAAATCTC TCGAAACGCT

CTCGAAGTTC

CAAATTTTCT CTTAGCATTC TCTTTCGTTT CTCGTTTGCG TTGAATCAAA

GTTCGTTGCG

ATGGCGGATG TTCAGATGGC TGATGCAGAG ACTTTTGCTT TCCAAGCTGA

GATTAACCAG

CTTCTTAGCT T 120 180 240 300 360 371 WO 97/46078 PCTIUS96/09453 146 INFORMATION FOR SEQ ID N0:25: SEQUENCE CHARACTERISTICS: LENGTH: 29 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID GGATCCGGAT CAAAAATGGG AAGGGGTAG 29 INFORMATION FOR SEQ ID NO:26: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO;26: GGATCCGCTG CGGCGAAGCA GCCAAGGTTG

Claims

1. A substantially purified" nucleic acid molecule encoding an API gene product having an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8.

2. A substantially purified nucleic acid molecule having a nucleic acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7.

3. A substantially purified nucleotide sequence having at least ten contiguous nucleotides of the nucleic acid molecule of claim 2, provided that said nucleotide sequence does not encode a portion of a MADS domain.

4. A vector, comprising a nucleic acid molecule encoding an API gene product selected from the group consisting of: the nucleic acid molecule of claim 1 and the nucleic acid molecule of claim 2. A method of producing an API gene product, comprising expressing a nucleic acid molecule selected from the group consisting of: the nucleic acid molecule of claim 1 and the nucleic acid molecule of claim 2.

6. A substantially purified API gene product having an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8.

7. An antibody that specifically binds the API gene product of claim 6. 148

8. The antibody of claim 7, wherein said antibody is a monoclonal antibody.

9. An expression vector, comprising a nucleic acid molecule encoding an API gene product having an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO:6 and SEQ ID NO: 8. The expression vector of claim 9, wherein said expression vector is a plant expression vector comprising a heterologous regulatory element.

11. The plant expression vector of claim wherein said heterologous regulatory element is a constitutive regulatory element.

12. The plant expression vector of claim 11, wherein said constitutive regulatory element is a cauliflower mosaic virus 35S promoter.

13. The plant expression vector of claim wherein said heterologous regulatory element is an inducible regulatory element.

14. A kit for converting shoot meristem to floral meristem in an angiosperm, comprising the plant expression vector of claim A kit for promoting early reproductive development in a seed plant, comprising the plant expression vector of claim