EP0640132A1 - A method for controlling and determining plant organ morphogenesis, a homeotic gene, a promoter element therefor, and related uses thereof - Google Patents

Abstract

A method for controlling the morphogenesis of plant organs is disclosed which comprises causing the underexpression of a homeotic gene responsible for determining the identity of the plant organ in object. The method has particular application to flowers, where, by way of a first example herein, the flower petunia has been modified to present an additional whorl of petals derived from the modification of sepals. The additional whorl of petals may be formed by the conversion of sepals to petals, and/or by the stimulation of the growth of the existing petal structures to define inverted structures that appear to present a second whorl. The hometoic gene green petal in petunia has been cloned and characterized and is presented herein, both alone and in association with expression control sequences and in vectors for transgenic ectopic expression. In a second example, the same flower may be modified to present a whorl of petals having sepaloid characteristics along at least a portion thereof. The invention extends to a promoter construct derived from the CaMV 35S promoter and designated herein m35S. The promoter is particularly suited for the construction of homeotic transgenes in accordance wtih the present method. Petunia flowers having a whorl of modified petals prepared by the present method are likewise disclosed.

Description

A METHOD FOR CONTROLLING AND DETERMINING PLANT ORGAN

MORPHOGENESIS, A HOMEOTIC GENE, A PROMOTER ELEMENT

THEREFOR, AND RELATED USES THEREOF

5 TECHNICAL FIELD OF THE INVENTION

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The present invention relates generally to the genetic determination of plant morphology, and more particularly, to the control of such morphology by techniques involving recombinant genetic technology. 10

BACKGROUND OF THE INVENTION

The study of plant morphology and morphogenesis has developed extensively with the advent of recombinant technology. Investigators have identified individual

15 homeotic genes that act either alone or in concert with each other to determine structures in the developing plant. Variations in plant morphology have been noted through the ages, and recent studies have revealed that mutations of critical homeotic genes may account for such structural aberrations. The bulk of these investigations have sought to determine both the manner in which such aberrations

20 result and possible avenues for application of such aberrations either to develop new plant strains and structures.

More particularly, investigations have focused on the morphological determination of flowers, as represented by the following publications [Sommer et al. EMBO J.

25 9(3):605-613 (1990); Schwarz-Sommer et al. , SCIENCE 250:931-936 (1990); Schwarz-Sommer et al., EMBO J. 7(l):251-263 (1992); and Jack et al., CELL, 68:683-697 (1992)]. The noted publications all deal with the elucidation of homeotic genes, such as the gene deficiens found in Antirrhinum majus (snapdragon) and the gene APETALA3 found in Arabidopsis thaliana.

30 Specifically, gene sequences are disclosed as are aspects of gene structure that control differentiation (the MADS-box), and their interaction with other homeotic genes determining flower organ morphology. These investigations identified the mutation of the genes and the consequences of such mutation, and sought to better understand the interrelationship of the combinatorial actions between the homeotic genes determining the four primary components of the flower.

SUMMARY OF THE INVENTION

The present invention is predicated in part on the broad discovery that the controlled expression of a homeotic gene, such as the gene green petal disclosed herein, can cause the conversion of a particular plant organ to another desired plant organ. More particularly, the invention is illustrated in a first embodiment by the overexpression of the homeotic gene green petal in petunia, to cause the conversion of sepals to petals so that an additional whorl of petals results. A further observation in this embodiment is that, in some instances the wild type petals exhibit lateral growth and form inverted structures that give the appearance of an additional whorl of petals.

In a second embodiment of the invention, the overexpression of the same transgene causes the petal structures of the petunia flower to exhibit varying degrees of sepaloid characteristics. This modification is believed to occur by causing the downregulation and consequent underexpression of the homeotic gene green petal in wild type petunia plants.

The concepts of introducing a homeotic transgene and by so doing, either inducing overexpression of homeotic genes in plants, or selectively suppressing the expression of the same gene in the wild type plant, in both instances to cause structural changes in plant organs as demonstrated herein has never before been considered or attempted. It is these concepts and their effectuation in the present invention that constitutes a further elucidation and development of the broader discovery of morphological control through control of homeotic gene expression, and that poses a significant opportunity for the development of controlled structural determination in plant organs. More particularly, the present invention relates to the modification of the organs of a flower to modify the structure of the wild type petals without modification to the reproductive portions thereof. For example, the underexpression by mutation of certain homeotic genes has been observed to cause the conversion of the reproductive organs to petals [Coen, E.S., ANNU. REV. PLANT PHYSIOL. PLANT MOL. BIOL. 42:241-279 (1991); Coen, E.S., et al., NATURE 253:31-37 (1991)]. However, this type of expression has not been obtained by genetic engineering. Also, this type of flower presentation is naturally more limited in view of the compromise to reproductive capability that is involved. It is more desirable in some instances to produce a more attractive flower that retains all of its reproductive capabilities.

Likewise, the elimination of gene expression in a plant by a phenomenon known as co-suppression has been previously proposed [van der Krol, A.R., et al., THE PLANT CELL 2:291-299 (1990); Napoli, C, et al., THE PLANT CELL 2:279- 289 (1990)]. Both publications deal exclusively with the expression of genes involved with pigmentation and as none of the experiments involved the modification of the expression of genes responsible for organ determination, the results and conclusions presented in these publications are of limited relevance to the present invention.

In a first particular aspect, a petunia flower is converted to define an additional whorl of petals by the ectopic expression of the homeotic flower gene green petal that is itself the subject of a further aspect of the present invention. The overexpression of the green petal gene causes the conversion of sepals to petals, and is also found in some instances to stimulate lateral growth of the existing petals to form inverted petal structures that give the appearance of the formation of an additional whorl of petals. Further, the inverted petal structures may come into contact and fuse to each other, and diverge from the existing petal structure. The invention also extends to the homeotic flower gene green petal in petunia having the DNA sequence set forth in Figure 2 and in Sequence Identification No. 1 (SEQ ID NO:l) presented in co-parent application Serial No. 07/867,580 filed April 13, 1992, and later on herein.

In the second particular aspect, the petunia flower is converted to define petals with sepaloid characteristics by introducing a transgene construct corresponding to the green petal gene of the present invention. The introduction of this green petal clone is observed to suppress the expression of the target endogenous gene and as demonstrated herein, causes this conversion of the petal structure.

The invention extends further to DNA sequences that code the expression of the amino acid sequence set forth in Figure 2, as well as DNA sequences that would hybridize thereto. The invention further extends to the operative linkage of said DNA sequence to an expression control sequence, including promoters such as the 35S promoter of the cauliflower mosaic virus. In a particular embodiment of the invention, the expression control sequence may be the modified 35S promoter (m35S) disclosed herein. Naturally, other promoters such as the 35S promoter represented by VIP27 depicted in FIGURE 7, nopaiine synthetase and the rbcs E9 gene from pea are representative of other nonlimiting examples thereof. The CaMV 35S promoter is preferred as it is constitutively expressed and is particularly well-suited for the method illustrated herein.

Likewise, the invention extends to a plant transformation vector containing the DNA sequence of Figure 2, and specifically, to the pMON 530 vector illustrated in the restriction map presented in Figure 3 herein. The invention also extends to a novel promoter construct for the homeotic flower gene green petal in petunia designated herein VIP 149 and depicted in FIGURE 5 A. The promoter of the invention is comprised of the -90 to +8 fragment of the CaMV 35S promoter and a fragment corresponding to four ligated fragments comprised of two alternations of each of the DNA sequences denominated 4xB3 and 2xTX4, respectively, set forth in FIGURE 5B and in respective Sequence Identification Nos. 2 and 3 herein, corresponding to SEQ ID NO:l and SEQ ID NO:2 presented in co-parent application Serial No. 07/909,589 filed July 6, 1992.

Accordingly, it is a principal object of the present invention to provide a method for controlling the structural determination of plant organs.

It is a further object of the present invention to provide a method as aforesaid that may be practiced with a high degree of accuracy and reproducibility.

It is a still further object of the present invention to provide a method as aforesaid which involves the overexpression of a homeotic gene.

It is a still further object of the present invention to provide a method as aforesaid which involves the co-suppression of a homeotic gene.

It is a still further object of the present invention to provide a method as aforesaid wherein flowers may be modified to define additional whorls of petals without affecting the size or function of any reproductive structures defined by the wild type thereof.

It is a still further object of the present invention to provide a method as aforesaid wherein flowers may be modified to define petals with sepaloid structural characteristics.

It is a still further object of the present invention to define a cloned homeotic flower gene for the flower petunia.

It is a still further object of the present invention to employ the homeotic flower gene for petunia as aforesaid in a method to produce a petunia flower having instead of sepals an additional whorl of petals.

It is a still further object of the present invention to define a promoter element for the expression of a homeotic flower gene for the flower petunia.

Other objects and advantages will become apparent to those skilled in the art from a review of the ensuing description which proceeds with reference to the following illustrative drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGURE 1 illustrates the interaction of the homeotic genes defining the structures of the flower, with Figure IA depicting schematically the expression of each of the component genes to define the structures indicated below, and Figure IB illustrating the present invention wherein the overexpression of one of the genes (i.e., a component of "B") interacts with the gene referred to as "A" to cause the conversion of sepals to petals, as specifically illustrated herein. The model of Figure IA was presented in Coen et al., 1991, supra.

FIGURE 2 depicts the full nucleic acid sequence for the homeotic gene green petal as well as the deduced amino acid sequence thereof. The nucleotides are numbered from 1 to 881, and the amino acids are numbered from 1 to 231. This sequence is identically depicted in the SEQUENCE LISΗNG presented later on herein, in accordance with 37 C.F.R. 1.821-825, enacted October 1, 1990, and is cumulatively and alternately referred to as SEQ ID NO:l.

FIGURE 3 is a restriction map illustrating the preparation of the' vector containing the green petal gene that was prepared for the practice of the present method in accordance with the first aspect of the invention.

FIGURE 4 is a restriction map illustrating one aspect of the preparation of the vector containing the promoter element of the present invention, and particularly, the combination of the cDNA of the green petal gene with the vector VIP149. FIGURE 5A is a restriction map illustrating the construction of the promoter of the present invention.

FIGURE 5B depicts both the upper and lower strands of nucleic acid sequences for the fragments identified herein as 4xB3 and 2xTX4, respectively, that are included in the promoter element of the present invention. The nucleotides for 4xB3 are numbered from 1 to 252, and the nucleotides for 2xTX4 are numbered from 1 to 114. Both sequences are identically depicted in the SEQUENCE LISTING presented later on herein, in accordance with 37 C.F.R. 1.821-825, enacted October 1, 1990, and are referred to as SEQ ID NO:2 and SEQ. ID NO:3, respectively.

FIGURE 6 is a restriction map illustrating a further aspect of the preparation of the vector containing the promoter element of the present invention, and particularly, the introduction of the vector containing the combination of the cDNA of the green petal gene and the m35S promoter into the vector pMON721 to form VIP162.

FIGURE 7 is a restriction map illustrating the preparation of an alternate vector used in the method of the present invention, and particularly, the construction of the vector VIP152.

FIGURE 8 is a restriction map illustrating the construction of the combination of the cDNA of the green petal gene and that of the glucuronidase coding sequence (GUS) to form the construct identified as V1P142, the operative portion of which in turn, is ultimately introduced into VIP27 to form the vector VIP 152.

FIGURE 9 is a photograph depicting the appearance of a petunia flower having a phenotype prepared by the first aspect of the method of the invention.

FIGURES 10A-10D are a series of photographs depicting the appearance of a petunia flower having a variety of phenotypes prepared by the second aspect of the method of the invention.

FIGURE 11 depicts restriction maps of the chimeric gene constructs J84, VIP 162 and VIP 186 prepared in accordance with Example 3 herein.

FIGURE 12 comprises schematic diagrams of the Petunia hybrida wild type flower

(V26).

Left: A longitudinal section illustrating the fusion of the stamen filaments to the corolla tube and the faces on which trichomes can be detected. CF (congenital fusion) indicates the region where the stamen filament is fused to the corolla tube.

Se = Sepal, Li = Corolla Limb, Tu = Corolla Tube, An = Anther, St = Style and Stigma, Ov = Ovary.

Right: Floral diagram. Se = Sepal, Pe = Petal, St = Stamen, Ca = Carpel

FIGURE 13 depicts the petunia V26 flower.

(A) A V26 flower.

(B) Two sepals from the first whorl. Note that the sepals are fused at the base which contains less chlorophyl. (C) A longitudinal cut flower bud with two sepals removed. Note the stamen filaments (Fi) which are fused near the base to the corolla tube (Tu) and the trichomes at the outer face of the corolla tube.

(D) A stained cross section of a V26 flower bud, near the base, showing the fusion of the filaments to the corolla tube. (E) Close-up of a stamen filament near the anther.

(F) An upper epidermal peel from the first whorl sepal tissue.

(G) An epidermal peel from the adaxial face of the second whorl tube tissue. (H) An upper epidermal peel from the second whorl petal limb.

(I) A lower epidermal peel from the second whorl petal limb, between the main veins.

(J) A lower epidermal peel from the second whorl petal limb, near the main vein. Tu = tube, Se = sepal, An = anther, Fi = filament, St = style, Ov = ovary, Vertical bar, 1 cm; horizontal thick bar, 1 mm; horizontal thin bar, 0.1 mm.

FIGURE 14 depicts sections of V26 and gp (PLV) flowers. (A) A longitudinal section of a V26 inflorescence. The numbers indicate the whorl, L = leaf

(B) A longitudinal section of a gp (PLV) inflorescence. The numbers indicate the whorl.

(C) A transverse section of a V26 flower. (D) A transverse section of a gp (PLV) flower. Note that the stamen filaments are not fused to the second whorl sepal-tube (c.f. Figure 2D).

(E) Close-up of a transverse section of a V26 flower showing sepal and petal tissue.

(F) Close-up of transverse section of a gp (PLV) flower, showing first and second whorl sepals.

Numbers refer to the floral whorl. L = Leaf, Se = Sepal, Pe = Petal, St = - Style, Ov = Ovary, An = Anther, Fi = Filament. Vertical bar, 1 cm; horizontal thick bar, 1 mm; horizontal thin bar, 0.1 mm.

FIGURE 15 depicts the flower of gp (PLV).

(A) A mature gp (PLV) flower.

(B) Two sepals from the first whorl. Note that the sepals are fused near the base.

(C) Two sepals from the second whorl. Note that the sepals are fused for half of their length. (D) An upper epidermal peel from the second whorl sepal.

(E) A lower epidermal peel from the second whorl sepal.

(F) A longitudinal section of a mature gp (PLV) flower. Note that the stamen filaments are not fused to the second whorl sepals.

(G) A sepaloid stamen often found in gp (PLV) flowers. (H) A gp (PLV) flower at a late stage of development. Note that the extra third whorl sepaloid organs can develop regions with petaloid characteristics (arrow). (I) Close-up of gp (PLV) flower with the first and second whorl sepals removed. Note the stamen filaments (not fused to the second whorl organs) and a sepaloid sixth organ, initiated between the stamen (arrow).

(J) Close-up of a gp (PLV) stamen filament, showing petaloid cells and trichomes. Vertical bar, 1 cm; horizontal thick bar, 1 mm; horizontal thin bar, 0.1 mm. For abbreviations see legend to Figure 13.

FIGURE 16 presents the results of the expression of MADS-box genes and CAB in floral organs of V26, gp (PLV) and co-suppression plants. Total RNA was isolated from young and mature flower buds of V26 (upper panels), PLV (middle panels) and SD15c (lower panels). Filters containing 7 μg of RNA per lane were hybridyzed to specific probes derived from the genes indicated. (A) pMADSl, (B) pMADS2, (C) fbpl, (D) pMADS3, (E) pMADS4, (F) CHS, (G) CAB. The panels within one box were derived from one filter, therefore the strength of the hybridization signal can be directly compared within a box. Lane 1, 2, 3 and 4 represent RNA isolated from whorl 1, 2, 3 and 4, respectively. (H) Total RNA - was isolated from young and mature flowers of transgenic line SD15d, Lane 2/3 represents RNA isolated from the combined second and third whorl tissue. Lane 1 and 4 represent RNA isolated from whorl 1 and 4, respectively.

FIGURE 17 presents Southern blot analysis of wild type and gp (PLV) genomic DNA. Genomic DNAs were digested with Hindiπ, size fractionated on an agarose gel, and blotted onto a Genescreen Plus filter. The blot was hybridized to a full length pMADSl cDNA (see Methods) and after hybridization it was washed under high stringent conditions. The three pMADSl gene fragments are indicated by arrows. Lane 1, V26; lane 2, V30; lane 3, W115; lanes 4-7, a segregating population of wild type and gp (PLV) plants; lane 4 and 6, wild-type plants; lane 5 and 7, gp (PLV) plants.

FIGURE 18 presents the results of phenotypic analyses of flowers of pMADSl complementation plants. Leaf tissue from hybrid GP/gp was used for transformation with the 35S-pMADSl gene construct J84 (see Methods, Example 3) and one transgenic, line (Ml) which showed an over-expression phenotype (see Halfter et al., 1993) was back-crossed with gp (PLV). Genomic DNAs of progeny plants (Mla-z) were analyzed for the presence of wild-type pMADSl and the 35S-pMADSl transgene. Flowers of plants that did not carry the wild-type pMADSl gene but contained one or more copies of the 35S-pMADSl transgene (Mla-d) were analyzed (B-F).

(A) The transformed GP/gp plant which showed an overexpression phenotype (Ml). This plant was crossed with gp (PLV) and Mla-d are some of the progeny plants.

(B) Close-up of a Mlb flower, showing a gp phenotype but has small sectors of petal tissue in the second whorl.

(C) and (D) a flower on Mlb at a late stage of plant development, before and after anthesis. Note the petal tissue that has developed after an thesis. (E) Mlc, heterozygous for the 35S-pMADSl transgene (Southern analysis not shown).

(F) Mid, homozygous for the same 35S-pMADSl transgene insert (Southern analysis not shown).

(G) Second whorl of Mlb, showing the partial petal development. (H) Close-up of second whorl of Mlb. Note the trichomes on the adaxial face of the petal.

(I) A corolla of Mlc. Note that the sepal structure can still be recognized through the corolla tissue, indicating that most of the corolla limb is derived from the second whorl (default) sepal. (J) Flower buds of V26

(K) Flower buds of Mlc

(L) Inside of a V26 second whorl corolla tube. Note the absence of trichomes on the adaxial face.

(M) Inside of a Mlc second whorl corolla tube. Note the presence of trichomes on the adaxial face.

(N) A petaloid stamen of Mlc Vertical bar, 1 cm; horizontal thick bar, 1 mm; horizontal thin bar, 0.1 mm.

FIGURE 19 presents Northern blot analysis of gp transgenic plants expressing pMADSl or pMADS2. Total RNA was isolated from mature flower buds of gp (PLV) transgenic plants expressing the 35S-pMADSl transgene J84 (A) or the 35S-pMADS2 transgene VIP186 (B). Equal amounts (7 μg) of RNA were analyses on identical Northern blots using gene-specific probes. Lane 1, 2, 3 and 4 represent RNA isolated from whorl 1, 2, 3 and 4, respectively. The signal for pMADS2 and fbpl in Figure 19B can be compared directly to the signal in Figure 16B and 16C, respectively (same hybridization)

(A) in Ml(a) the expression of the 35S-pMADSl transgene is very low and can only be detected in the third and fourth whorl upon prolonged exposure (not shown). In these transgenic plants the expression of pMADS2 and fbpl is mainly in the mature third whorl. In Ml(c+d) the average expression of the 35S-pMADSl transgene is higher in all four whorls. In these plants the expression of pMADS2 and fbpl is up-regulated in the second whorl, compared to non-transformed gp plants (c.f. Figure 16B and 16C).

(B) Transgenic gp plants that express the 35S-pMADS2 transgene (Ml). Tissue from five indepenent trangenic flowers was combined for northern analysis. The average expression of pMADS2 in the first, second and fourth whorl is low

(presumably from the transgene) and has no effect on the expression of fbpl (c.f. Figure 16C)

FIGURE 20 presents the results of phenotypic analysis of pMADSl co-suppression plants. Wild-type petunia plants (V26) were transformed with a 35S-pMADSl chimeric gene construct (VIP 162). Transgenic plant SD15 was selfed and flowers of progeny plants (SD15a-d), showing different degrees of co-suppression were analyzed.

(A) SD15a. Although the effect on second whorl petal development is mild, the third whorl stamen filaments are not fused to the petal tube of this flower.

(B) SD12. The tube and petal tissue of this flower are not fully developed. (C) SD15b. The second whorl tube is much reduced and in the limb sectors of petal or petaloid tissue have developed.

(D) SD15c. The flowers from this line are an almost completely phenocopy of gp (PLV) flowers. (E) SD15c. Same flower as (D), but one week later. After anthesis the second whorl can develop some petaloid characteristics.

(F) Inside of a SD15b flower. Part of the first and second whorl organs were removed to show that the stamen filaments are not fused to the second whorl organs and that extra, third whorl sepaloid organs develop in this flower (indicated by arraw).

(G) Second whorl sepaloid structure of SD3, showing sectors of petal tissue.

(H) Close-up of second whorl sepaloid structure of SD3, showing sectors of green cells, white cells and cells pigmented with anthocyanins.

(1) and (J) An epidermal peel from SD3 second whorl tissue (see H), showing small, fuUy-pigmented 'petal' cells next to non-pigmented and slightly pigmented jigsaw-shaped epidermal cells.

(K) Close-up of a stamen filament of a SD3 flower, showing petaloid cells and trichomes.

(L) SD15d. Most of the second whorl in these flowers does not develop or is fused to the stamens.

- (M) SD15d. All of the organs of the second/third whorl that are produced in one flower.

Vertical bar, 1 cm; horizontal thick bar, 1 mm; horizontal thin bar, 0.1 mm.

FIGURE 21 presents the different end-stages of petal development in petunia. (A) Hand-made tissue sections from mature second whorl organs of:

(1) gp (PLV) sepal. The upper and lower epidermal cell layer are translucent, trichomes are present on both faces and the inner parenchyma cells are green.

(2) pMADSl co-suppression (SD15b). The upper and lower epidermal cell layer shows sectors of petaloid cells, pigmented with anthocyanins.

(3) pMADSl co-suppression (SD12). There is a reduction in trichome number in larger patches of petaloid epidermal cells. The parenchyma cells show only weak green pigmentation. ,

(4) pMADSl complementation (Mlc). The upper epidermal cell layer is almost completely petaloid, with only a few trichomes on the adaxial face. The inner parenchyma cells are small although still pigmented green.

(5) V26 petal. The upper and lower epidermal cell layer consist of small cone shaped cells, pigmented with anthocyanins. The parenchyma cells are small and white.

(B) A schematic presentation of the different end-stages of petal development in petunia. The default state of the second whorl organ is sepaloid, characterized by jigsaw-shaped epidermal cells, trichomes, stomata and large, green parenchyma cells. Petal differentiation suppresses the formation of trichomes, and stomata, while the epidermal cells can become pigmented and loose their characteristic jigsaw-shape (more round). Full petal differentiation results in small, white parenchyma cells and epidermal cells that are small, cone-shaped and pigmented purple with anthocyanins.

(C) A schematic presentation of the growth stages that transform a sepaloid organ into a petal. The sepal is formed by growth S. When petal differentiation is activated this growth is transformed into C and additional growth occurs (Cl, C2 and C3), leading to the petal stucture. FI is the growth of the filament which occurs above the zone of stamen initiation. The growth of C3 and F2 occurs under the zone of petal and stamen initiation, resulting in a congenital fusion of stamen filament to the corolla tube.

DETAILED DESCRIPTION

In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Maniatis, Fritsch & Sambrook, "Molecular Cloning: A Laboratory Manual" (1982); "DNA Cloning: A Practical Approach," Volumes I and II (D.N. Glover ed. 1985); "Oligonucleotide Synthesis" (M.J. Gait ed. 1984); "Nucleic Acid Hybridization" [B.D. Hames & S.J. Higgins eds. (1985)]; "Transcription And Translation" [B.D. Hames & S.J. Higgins, eds. (1984)]; "Immobilized Cells And Enzymes" [IRL Press, (1986)]; B. Perbal, "A Practical Guide To Molecular Cloning" (1984).

The amino acid residues described herein are preferred to be in the "L" isomeric form. However, residues in the "D" isomeric form can be substituted for any L- amino acid residue, as long as the desired functional property of immunoglobulin- binding is retained by the polypeptide. NH2 refers to the free amino group present at the amino terminus of a polypeptide. COOH refers to the free carboxy group present at the carboxy terminus of a polypeptide. In keeping with standard polypeptide nomenclature, J. BIOL. CHEM. , 243:3552-59 (1969), abbreviations for amino acid residues are shown in the following Table of Correspondence:

Q Gin glutamine

E Glu glutamic acid

W Trp tryptophan

R Arg arginine D Asp aspartic acid

N Asn asparagine

C Cys cysteine

It should be noted that all amino-acid residue sequences are represented herein by formulae whose left and right orientation is in the conventional direction of amino- terminus to carboxy-terminus. Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino-acid residues. The above Table is presented to correlate the three-letter and one-letter notations which may appear alternately herein.

If appearing herein, the following terms shall have the definitions set out below.

A "replicon" is any genetic element (e.g., plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo; i.e., capable of replication under its own control.

A "vector" is a replicon, such as plasmid, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment.

A "DNA molecule" refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in its either single stranded form, or a double- stranded helix. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA, such as that found in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes. In discussing the structure of particular double-stranded DNA molecules, sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA).

In accordance with the present invention and as stated earlier, a method is disclosed for the control and determination of plant organ identity, comprising the overexpression of one or more of the homeotic genes that have been determined to control the structural identity of the plant organs in question. The present invention is specifically illustrated herein with respect to the flower petunia, wherein the overexpression of the homeotic transgene green petal, results in a first embodiment in the conversion of sepals to petals, and in certain instances, the stimulation of petal growth to cause the formation of inverted petal structures from the extended lateral edges of the original petals. In a second embodiment, the overexpression of the green petal gene results in the conversion of petals to sepals. More particularly, the method of the invention appears to cause the down regulation of the expression of both the transgene and its endogenous counterpart, and it is this underexpression that is believed to cause in turn, the structural modification of the petals.

At present the invention extends further to the petunia flowers modified by the methods hereof, and to the cloned and characterized gene depicted in Figure 2 and in SEQ ID NO: 1 herein. The present invention arose from an extension of the genetic and molecular biological analysis of mutants. Genetic and molecular biological analysis of mutants of Arabidopsis and Antirrhinum has led to the proposal of a model for the action of homeotic genes to define flower organ identity. Three gene products have been identified to determine by individually or in a combinatorial manner, the flower organs (FIGURE 1, generally). The Antirrhinum gene ovulata specifies sepal development, but in combinatorial expression with the deficiens gene, petals are defined. The plena gene product is necessary for the development of the generative organs, the stamen and the carpel. While it is sufficient for carpel identity, additional gene expression of deficiens is required for stamen development.

It is a general precept of the invention, that, while particular homeotic genes may be present and necessary for the determination of particular organ structure, it is the sufficiency of their presence that can actually control structural determination. It is to this principle, therefore, that the present invention and that of the parent applications referred to herein, have been directed.

The practice of the present method is largely reliant on known techniques in molecular biology. That is, the expression of the gene in object may proceed by the preparation of the gene to include one or more promoters and the placement of the same in a suitable vector. In accordance with the first embodiment of the invention and as illustrated herein with reference to Figure 3, the cauliflower mosaic virus (CaMV) 35S promoter is employed within the plant transformation vector pMON 530. Specifically, the green petal cDNA was cloned into a λ Zap subcloned as an Smal fragment containing the complete coding region of the gene, a small part of the leader from the poly A tail and additional polylinker sequences from the cloning vector. In the second embodiment of the invention illustrated in FIGURES 4-8, variations of the cauliflower mosaic virus (CaMV) 35S promoter are employed within the plant transformation vector pMON721. Specific protocols for the preparation of vectors containing green petal cDNA are described in the Materials and Methods section of Examples 1 and 2 presented later on herein. These preparations are presented herein as illustrative and not restrictive, the scope of the invention naturally extending to other vectors, promoters and protocols suitable for use in plant transformation.

Promoters which are known or are found to cause transcription of RNA in plant cells, and particularly, promoters that show expression in the second whorl of flowers, have been described in the literature and can be used in the present invention. Such promoters may be obtained from plants or viruses and include, but are not limited to, the green petal (GP) gene, the nopaline synthase (NOS) and octopine synthase (OCS) promoters (which are carried on tumor-inducing plasmid of Agrobacterium tumefaciens), the wheat histone H3 gene, the rbcs E9 gene from pea, the cauliflower mosaic virus (CaMV) 19S and 35S promoters [Odell et al., NATURE 313:810 (1985)], the light-inducible promoter from the small subunit of ribulose bisphosphate carboxylase (ssRUBISCO) as set forth in commonly assigned U.S. Patent No. 4,990,607, and the mannopine synthase promoter [Velten et al. EMBO J. 3:2723-30 (1984); and Velten et al., NUCLEIC ACID RES. 13:6981-98 (1985)] and where desired, mixtures of one or more of the above. All of these promoters have been used to create various types of DNA constructs which have been expressed in plants, (see, e.g., PCT publication WO84/02913) (Rogers et al., Monsanto).

As mentioned above, the CaMV 35S promoter is preferred herein because of its ability to be constitutively expressed. More generally, the strength and specificity of the promoter will affect the degree of penetrance of the gene, and in turn, the extent of conversion of the sepals to petals, and the extent of the enhancement of petal growth. Accordingly, the selection and use of a promoter that is highly active (eg. exhibits strong expression) in sepals, and/or the use of multiple promoters, would further improve the results obtainable by the present method. Also, variations of this promoter and others may be included, i.e., promoters derived by means of ligation with different regulatory regions, random or controlled mutagenesis, strengthened promoters prepared by the addition of multiple and/or copies enhancer elements or the like as described by Kay et al. [SCIENCE 136:1299-1302 (1987)] and as specifically disclosed and claimed herein with respect to the construction of the m35S promoter of the present invention, and the invention is submitted to extend thereto.

Accordingly, and as set forth earlier herein, the invention extends to a particular promoter designated herein VIP149 and depicted in FIGURE 5A. The promoter of the invention is comprised of the -90 to +8 fragment of the CaMV 35S promoter and a fragment corresponding to four ligated fragments comprised of two alternations of each of the DNA sequences denominated 4xB3 and 2xTX4, respectively, set forth in FIGURE 5B and in respective Sequence Identification No. 2 (SEQ ID NO:2) and Sequence Identification No. 3 (SEQ ID NO:3).

While the DNA sequence of the homeotic gene green petal or other homeotic gene of interest may be embodied in a vector such as disclosed herein in its form as sequenced, it is to be understood that, where desired, such coding sequence may be modified, to create mutants, either by random or controlled mutagenesis, using methods known to those skilled in the art. Accordingly, the invention may extend to the expression of truncated proteins and fusion proteins, sense and antisense constructs, as well as unmodified genes.

The 3' non-translated region contains a polyadenylation signal which fimctions in plants to cause the addition of polyadenylate nucleotides to the 3' end of the mRNA. Examples of suitable 3' regions are the 3' transcribed, non-translated regions containing the polyadenylated signal of genes from the T-DNA of Agrobacterium, the soybean storage protein genes, the small subunit of the RuBP carboxylase gene, and the rbcs E9 gene from pea.

The RNA produced by a DNA construct of the present invention also contains a 5' non-translated leader sequence. This sequence can be derived from the promoter selected to express the gene, and can be specifically modified so as to increase translation of the mRNA. The 5' non-translated regions can also be obtained from viral RNAs, from suitable eukaryotic genes, or from a synthetic gene sequence. The non-translated leader sequence can be part of the promoter sequence, or can be derived from an unrelated promoter or coding sequence as discussed above.

A DNA construct of the present invention can be inserted into the genome of a plant by any suitable method. Suitable plant transformation vectors include those derived from a Ti plasmid of Agrobacterium tumefaciens, such as those described by Herrera-Estrella et al. [NATURE 303:209 (1983); Bevan et al. NATURE 304: 184 (1983); Klee et al. BIO/TECHNOLOGY 3:637-642 (1985); Fraley et al., BIOTECHNOLOGY 3:629 (1985) and EPO Publication 120,516 (Schilperoort, et al.)]. In addition to plant transformation vectors derived from the Ti or root- inducing (Ri) plasmids of Agrobacterium, alternative methods can be used to insert the DNA constructs of this invention into plant cells. Such methods may involve, for example, the use of liposomes, electroporation, chemicals that increase free DNA uptake, transformation using viruses or pollen and the use of microprojectiles.

Choice of methodology for the regeneration step is not critical, with suitable protocols being available for hosts from Leguminosae (alfalfa, soybean, clover, etc.), Umbelliferae (carrot, celery, parsnip), Cruciferae (cabbage, radish, rapeseed, etc.), Cucurbitaceae (melons and cucumber), Gramineae (wheat, rice, corn, etc.), Solanaceae (potato, tobacco, tomato, peppers) and various horticultural crops. Gene expression can be altered in plants from each of the aforesaid families pursuant to the present invention.

The present invention will be better understood from a consideration of the following examples which are presented for purposes of illustration and not by way of limitation. It is clear from the present disclosure, that a variety of structural modifications may be achieved by means of the principles set forth herein, and the invention is accordingly believed to extend to such diverse modifications.

EXAMPLE 1

As set forth earlier, the homeotic gene green petal from petunia was cloned and characterized. The following experiment was performed with this gene, and discloses the preparation of a petunia flower with two whorls of petals by transgenic ectopic expression of the gene in accordance with the method of the present invention.

Materials and Methods In order to isolate, the deficiens homologue from petunia, a probe spanning the conserved region of the so called MADS-box of the Antirrhinum deficiens gene was used (Sommer et al, 1990). A cDNA library was constructed using RNA derived from joung petunia flower buds. The library was screened with the described probe for genes containing the homologous MADS-box region. Sequence homology and organ specific expression in petal and stamen identified the obtained cDNA clone as a petal/stamen specific homeotic flower gene. Southern blot analysis had shown that this gene is not present in a mutant called "green petal", which was induced by gamma irradiation. In this mutant, the petal structures were converted to sepals by the absence of this gene; hence, this gene has been assigned the name "green petal".

In order to transform sepals into petals, artificial gene expression of the green petal gene in the first whorl of the petunia flower was performed. Accordingly, the cDNA (Smal-fragment) of the green petal gene was cloned into the Smal-site of the expression cassette of the plant transformation vector pMON 530 (Cuozzo et al., 1987). This vector ∞ntains the Cauliflower Mosaic Virus 35S promoter and the E9 polyadenylation site of the rbcs E9 gene from pea, which results in constitutive expression of the transgene in planta (Fig. 3). The recombinant vector was transferred to the Agrobacterium tumefaciens strain GV31111SE.

Transformation of petunia was performed by Agrobacterium tumefaciens mediated gene transfer using the leaf disc method (Horsch et al., 1985). Transgenic plants were selected on kanamycin and regenerated. Shoots, were rooted and transferred to soil in the greenhouse to score phenotypes.

Results The transgenic plants expressed of the green petal gene at high levels were analyzed by Northern blot experiments. The transgenic plants produced flowers with a homeotic phenotype. The sepals were partially converted into petals. At the position of sepals in the wild type flower, the transgenic flowers have a petal tube-like structure containing the petal color anthocyanin. In addition, the growth of existing petals was enhanced so that the petals grew laterally and inverted to present what appears to be an additional whorl of petals. In some instances the inverted portions of the petals fused to each other to form separate petal structures. Transgenic plants were selfed and FI progeny showed co-segregation of the transgene and the overexpression phenotype, confirming that the observed phenotype is stable and inherited to the next generations.

A flower representative of this phenotype is shown in Figure 4. Referring in detail to Figure 4, the illustrated phenotype exhibits the development of additional inverted petals resulting from the lateral outgrowth of the wild-type structures, as well as substantial conversion of the sepals into petal structures.

EXAMPLE 2

The following experiment illustrates the second aspect of the invention. In this Example, a petunia flower was prepared with petals having sepaloid characteristics by transgenic ectopic expression of the green petal gene in accordance with the method set forth below.

Materials and Methods

The green petal (GP) coding sequence was isolated from a petunia Wl 15 cDNA library using a PCR fragment spanning the so-called MADS-box region from the Antirrhinum Deficiens gene. The cDNA was cloned as an Xbal-Kpnl fragment in the vector VIP149 to generate clone VIP160 (see FIGURE 4). VIP149 contains the -90 to +8 fragment of the CaMV 35S promoter (A-domain, Benfey et al., 1990), 4 copies of an optimized ASF-1 binding site (Katagiri et al., 1989), and 4 copies of a B3 domain (Benfey et al., supra). FIGURE 5 A shows the construction of VIP149 and FIGURE 5B shows the sequence of 4xB3 and 2xTX4. As stated earlier, this modified promoter has been named the m35S promoter. The m35S promoter plus GP coding sequence was isolated from VIP 160 as a partial HindlH-Kpnl fragment and cloned in the Hindϋl-Kpnl site of the binary vector VIP26 (van der Krol and Chua, 1991) to generate clone VIP 162 (see FIGURE 6).

The additional vector VIP 152 was constructed by cloning the GP-cDNA as Xbal- ERV fragment into VIP27 to generate VIP142 (see FIGURE 7). VIP27 contains the β-Glucuronidase (GUS) coding sequence from plasmid pBIlOl.l (Clonetec, Palo Alto CA) in the vector pBSII (Stratagene). This puts 80% of the GP coding sequence in front of the Glucuronidase (GUS) coding sequence, however the two coding sequences are not in frame. The fused sequence was isolated from VIP 142 as an Xbal-ERl fragment and cloned into the Xbal-ERl site of the binary vector VIP26 (see FIGURE 8). Constructs VIP27, 142, 149 and 160 are all in the vector pBSII (Stratagene), and constructs 162 and 152 are in the binary vector pMON721 (Monsanto).

VIP162 and VIP152 were mobilized to the Agrobacterium tumefaciens strain ABI and transformation of petunia line V26 was done by standard leaf disc transformation method (Horsch et al., 1985). After selection on kanamycin transgenic petunia plants were grown under normal greenhouse conditions.

Results

The petunia GP gene was isolated from a petunia W115 flower bud cDNA library using as a probe a PCR fragment from the Antirrhinum Deficiens gene. The Deficiens gene has been shown to have a B-function in Antirrhinum flower development (Coen et al., 1991 and FIGURE 1). From this screen a petunia cDNA was isolated and sequenced. The protein encoded by the cDNA showed a high homology to the Deficiens protein. RNA transcripts encoded by the cDNA were found in petal and stamen tissue of petunia flowers suggesting a B-gene function of this gene, in petunia flower development. The gene was named Green Petal gene (GP-gene), after the mutant petunia "green petal" in which this gene is missing.

The GP cDNA was cloned downstream of the m35S-promoter (VIP162; see Material and Methods) or downstream of the 35S promoter, fused out of frame to the GUS coding sequence (VIP152; see Material and Methods) and by transforming a wild type petunia plant with these transgenes, plants were obtained in which the GP function in flowers is partially to completely repressed. It is theorized that this suppression of GP expression is the result of the co-suppression phenomenon discussed earlier herein. This assumption is supported by the measurement of RNA by northern blot analysis, the results of which show that in these plants, GP RNA steady state levels are greatly reduced in the second whorl compared to that in second whorl tissue from untransformed plants (van der Krol, unpublished).

FIGURE 10A-D are photographs taken of three transgenic plants that show an increasing loss of GP function during petal development. Transgenic plant VIP162-15 (FIGURE 10A) shows a reduction of the corolla pigmentation and a slightly reduced corolla outgrowth. FIGURE 10B shows the flower of a plant with a more severe effect on petal development. The petal shows reduced growth in sectors; some sectors are green, some are white and some sectors are pigmented (transgenic VIP162-3, FIGURE 10B).

Detailed analysis of these petals also shows that trichromes are present on the surface of the green and white petal tissue. This again indicates a partial conversion of the petal into sepal since normally trichromes are found on both surfaces of the sepal but not on the upper surface of the petal. The most severe suppression of GP function results in a phenocopy of the mutant "green petal" phenotype (transgenic VIP152-1, FIGURES IOC and 10D). The petals are converted to sepals and show upon aging mild pigmentation at the outer edges.

EXAMPLE 3

This example presents the text of supplemental studies conducted by the inventors herein that confirm the activity of the homeotic gene green petal. The results presented herein refer to a gene identified as pMADSl, which corresponds to the green petal gene of the present invention.

Accordingly, the 'wild-type' petunia flower and the flower of the homeotic mutant gp (line PLV) are described below, and it is shown that the latter contains a deletion for the petunia MADS-box gene pMADSl. Moreover, the role of pMADSl in second whorl petal and third whorl stamen development is demonstrated, through both complementation and co-suppression studies. With these experiments a series of flowers with different end points of second whorl petal developmentwas created, thus enabling the dissection of the differentiation pathway for the conversion of a sepaloid structure into a petal. By analyzing the expression patterns of five MADS-box genes from Petunia hybrida during wild-type and mutant flower development, the question was addressed as to why gp (PLV) is mainly affected in one whorl. The following studies indicate that the components that are used for floral development are similar in different plant species, but their different interactions may lead to differences in floral organ determination.

Methods

Plant material and transformations.

Petunia plants were grown under standard greenhouse conditions. The pMADSl and pMADS2 cDNA's were isolated from Petunia hybrida line W115. The fbpl gene which has been isolated and described by Angenent et al. (1992) was isolated from Petunia hybrida line R27. The petunia 'green petal' mutant line PLV was resulted from gamma-ray treatment and was kindly provided by Dr. E. Farcy (INRA, Dyon, France). Other hybrid lines used in our experiments were Petunia hybrida line V26 and V30. The (presumed) ancestor petunia lines that were used are: Petunia axillaris (SI and S2, the different S numbers designate different origins), Petunia inflata (S6 and S14), Petunia parviflora (S4), Petunia violacea (S9 and S10), Petunia integrifolia (S12 and S13) and Petunia parodii (S8). These lines were kindly provided by Dr. R. Koes (Free University, Amsterdam, Holland). Plant transformations were performed as described by Horsch et al. (1985) using leafdiscs from V26 (construct VIP 162), a hybrid of V26 and PLV (construct J84) or PLV (construct VIP186).

DNA cloning strategies

The overexpression construct J84 was made by cloning the pMADSl cDNA fragment into a vector containing the CaMV 35S promoter and rbcS-E9 polyA addition signal (Halfter et al, 1993). The pMADSl co-suppression construct VIP 162 was made by cloning the cDNA as an Xbal -Kpn 1 fragment downstream - of a modified 35S (m35S) promoter in the vector pBSII (Stratagene) to generate clone VIP160. The modified 35S promoter contains the -90 to +8 fragment of the CaMV 35 S promoter (AS10, Benfey et al., 1990) with 4 copies of the B3 domain (Benfey et al., 1990) and 4 copies of an optimized AS-1 binding site (Katagiri et al, 1989) placed upstream. The m35S promoter has been shown to direct the expression of a β-Glucuronidase reporter gene in all cell layers of the petunia petal (van der Krol, unpublished results). The m35S promoter plus pMADSl coding sequence was isolated from VIP 160 as a partial Hindlll-Kpnl fragment and cloned between the Hindlll-Kpnl site of the binary vector VIP26 (van der Krol and Chua, 1991) to generate clone VIP162. The pMADS2 gene construct was made by inserting the EcoRl fragment of the pMADS2 cDNA into a binary vector which contains both the mCaMV-35S promoter and the rbcS-E9 polyA addition signal to form VIP 186. The chimeric gene constructs that were introduced into the petunia genome are set forth in Figure 11. Southern and Northern analyses.

Genomic plant DNA, isolated from about 1 gram of leaf tissue, was digested with restriction endonucleases, size fractionated on agarose gels and blotted onto Genescreen-plus membrane (DuPont). The hybridization and washing conditions were similar as for the Northern blots (see below). Total RNA was isolated from plant tissue using the RNaid isolation procedure (BIO 101). Flower buds were dissected into first, second, third and fourth whorl tissue. The young flower bud material we used measured, from base to the tip of the first whorl sepal, 5-15 mm for V26 and V30, 5-20 mm for W115 and 5-10 mm for PLV. In all lines, at this stage the second whorl tissue is mainly light green in color and covers the third and fourth whorl organs and the third whorl stamen filaments have not elongated. The mature flower bud material consisted for V26 and V30 of closed flower buds 5-6 cm long (measured from the base to the tip of second whorl petal), for W115 closed flower buds 6-7 cm long and for PLV open flowers with stamen filaments fully elongated, but before anthesis. For V30, V26 and W115 the mature floral bud stage coincides with the peak in the second whorl CHS gene expression (Koes et al., 1989).

Equal amounts (10 μg) of total RNA were fractionated on 1.2% agarose gels containing 6% formaldehyde. Gels were blotted onto Genescreen-plus (DuPont) according to the manufacturers instructions and hybridized to random primed labeled DNA (Boehringer) in 20% formamide, 5xSSC, 1% SDS, 5x Denhards, 10 μg/ml Salmonsperm DNA at 42°C. Blots were either washed at non-stringent conditions (0.5 hr., 2x SSC, 65°C) or stringent conditions (0.5 hr., 0.2x SSC, 65°C). Gene-specific probes (cDNA fragments without the MADS-box region) were used for each of the genes. The fbpl probe, covering nucleotides 494-760 (Angenent et al., 1992) was generated by polymerase chain reaction amplification using R27 genomic DNA as template.

Phenotypic analysis and imaging

The flower close-ups and hand-made tissue sections were photographed under a NIKON SMA-U stereomicroscope. Epidermal peels taken from sepal or petal tissue were, before photographing, vacuum infiltrated with water to remove air-pockets. The microscopic sections were made and stained as described by Natarella and Sink (1971) and photographed in bright field either under the NIKON SMZ-U stereo microscope or a NIKON optiphot microscope. All images were processed in Adobe photoshop and assembled in Aldus Pagemaker. The Northern images were compressed vertically (20%).

Results The wild type flower.

The flower development in petunia has been described in detail (Prior, 1957; Natarella and Sink, 1971; Sink, 1984; Turlier and Alabouvette, 1988). Because the transformations described in this paper were done in the Petunia hybrida line V26 we will give a short description of the essential features of the V26 flower for the purpose of comparison with mutant or transgenic flowers. The mature flower of V26 (Figure 12 and 13A) has in the first whorl five sepals that are fused at the base to form a calyx tube (Figure 13B). Regions in the calyx tube contain cells that make less chlorophyl as judged from the white color of the parenchyma cells. Growth of the corolla tube and of the filaments occur in part under the zone of interpetalous initiation, resulting in congenital fusion of the filaments to the corolla tube (Figure 12 and 13C). Figure 13D shows a stained cross-section near the base of a 10-mm long floral bud illustrating the fusion of the filament to the tube. From the point of separation the filaments become a smooth, round structure with flat, elongated epidermal cells that are lightly pigmented near the anther sacs (Figure 13E).

The lower and upper epidermal cell layer of the sepal appear similar morphologically, consisting of jigsaw-shaped epidermal cells, stomata and trichomes (Figure 13F). The inner and outer epidermal cell layer of the corolla tube are comprised of flat, elongated cells that may be pigmented (Figure 13G) and trichomes are only present on the outside of the corolla tube (see Figure 13C). On the limb, trichomes are only found on the lower epidermis and are mainly associated with the main vascular bundles. At the upper epidermal cell layer of the limb the cells are round, cone-shaped, and pigmented with anthocyanins (Figure 13H). By contrast, the epidermal cells at the lower side of the limb vary from jigsaw-shape to round (Figure 131 and 13J) and may have the characteristic cone-shape of the upper epidermal cells. Near the main vein the lower epidermal cells are not always pigmented (Figure 13J).

The 'green petal' fPL flower. The petunia gp mutant is characterized by a homeotic conversion of the second whorl petal into sepal. The gp phenotype was obtained in plants by a spontaneous mutation (line M64), by EMS treatment (line R100) and by gamma radiation mutagenesis (line PLV). All of these mutations are recessive. Here, the flowers of the gp line PLV are described (de Vlaming et al, 1984). Sections of young flower buds (up to 3-mm long, measured from the base to the tip of the first whorl sepals) of V26 and gp (PLV) are almost indistinguishable morphologically (cf. Figure 14A and 14B). In both V26 and gp (PLV) trichomes begin to develop on the abaxial face of the second whorl organs when the flower buds are about 2 -mm long. When the gp (PLV) floral buds are about 4-mm long the formation of trichomes can be detected at the adaxial face of the second whorl organs, whereas no trichomes are detected on the adaxial face of the V26 second whorl petal at this or any later stage of flower development. Figures 14C and 14D show a cross section through a 15-mm long flower bud of V26 and of gp (PLV), respectively. The parenchyma cells of the second whorl organ in gp (PLV) are not as large as those of the first whorl sepal but smaller than those of the petal, and the cell wall staining is more like that of sepal than petal (Figures 14E and 14F).

Figure 15 A shows the mature flower of gp (PLV). The first whorl sepals are fused at their base, as in V26 and show near the base only a very slight reduction in chlorophyl pigmentation as compared to the V26 sepals (compare Figure 15B with Figure 12B). The upper and lower epidermal cell layer of the gp (PLV) first whorl sepal are similar to those of the V26 sepal (not shown). The gp (PLV) second whorl sepals are slightly thinner than the first whorl sepal tissue and shows no marked reduction of chlorophyl synthesis near their base (Figure 15C). Figures 15D and 15E show an epidermal peel from the abaxial and adaxial face of the second whorl sepal, respectively. The epidermal cells on both faces resemble those of the V26 sepal (jigsaw-shaped cells, stomata and trichomes). Thus, the second whorl organs of gp (PLV) are sepals by virtue of their green pigmentation, cell size and shape, and the presence of trichomes and stomata on both faces.

Although stamen development in the gp (PLV) mutant is similar to that in wild-type petunia and leads to the formation of anther sacs which produce viable pollen, some developmental differences are apparent. In contrast to V26, the stamen filaments of gp (PLV) are not fused to the second whorl (Figure 15F, see also cross-section in Figure 14D). Sepalloid structures however, often emerge from the third whorl stamens (Figure 15G) or additional sepaloid third whorl organs are initiated between the stamen filaments (Figure 15H and 151). Upon maturation of the gp (PLV) flower (after anthesis has occurred) these green sepaloid structures develop regions with petaloid characteristics: cone shaped cells with anthocyanins (Figure 15H). Also, frequently petaloid cells and/or trichomes were detected on the filaments of the stamens (Figure 15J) which clearly differ - from the long, elongated epidermal cells found on wild-type stamen filaments (Figure 13E). The occurrence of the extra sepaloid structures and sepal/petaloid stamens in the third whorl may vary throughout the plant life cycle and may be influenced by growth conditions.

Four Petunia hybrida MADS-box genes.

The following describes the expression pattern in V26 and gp (PLV) flowers of four petunia MADS-box genes that were isolated in the laboratory of the inventors (Kush et al., 1993, Tsuchimoto, submitted) and fbpl, a petunia MADS-box gene isolated by Angenent et al. (1992). pMADSl has a 693-bp open reading frame encoding a protein which shows a 93% identity to the Antirrhinum DEFA MADS-box region and a 77% identity outside of the MADS-box region (Sommer et al., 1990). pMADS2 is 972-bp long and encodes a protein of 213 amino acids. This gene shares 87% identity with the Antirrhinum GLOBOSA gene MADS-box region and a 60% amino acid homology outside the MADS-box region (Trobner et al., 1992). fbpl also shares homology to GLO (87% identical within the MADS-box and 66% identical outside of the MADSbox). Both the fbpl and pMADS2 genes are present in the hybrid lines W115 (used to isolate pMADSl-4) and R27 (used to isolate fbpl) as well as in different presumed ancestor lines from petunia (see Methods; southern analysis not shown). The two other petunia MADS-box genes, pMADS3 and pMADS4, that were sequenced show homology to the Arabidopsis AGAMOUS gene (Yanofsky et al., 1990) and the AGL6 gene (Ma et al., 1991), respectively. Sequence analysis of these genes will be published elsewhere (S. Tsuchimoto, submitted).

MADS-box gene expression.

The expression pattern of the five petunia MADS-box genes described above was analyzed in different floral organs as well as in leaf, stem and root. It was found that pMADSl, 2 and 3 are expressed in the flower but not in vegetative organs, whereas pMADS4 is expressed in leaves as well as in flowers (Tsuchimoto, unpublished results). The expression pattern of fbpl in petunia has been reported - by Angenent et al. (1992). Initial experiments showed that the steady state mRNA levels in the different floral organs vary throughout development. Expression levels at early and late stages of development are therefor shown (for a description of stages see Methods). These initial studies also showed that the steady state mRNA levels of some pMADS-box genes may vary at similar stages of floral development among different lines, presumably reflecting the different rates at which floral organs mature in different genetic backgrounds. Two processes that are specifically associated with petal development are suppression of chlorophyl synthesis and enhancement of anthocyanin pigmentation in the petals. To monitor these events, the expression of the chlorophyll a/b binding protein (CAB) gene and the chalcone synthase (CHS) gene were also analyzed. For easy comparison we grouped for each of the MADS-box genes the expression profile in V26 (Figure 16A-16G, upper panels) and gp (PLV) (Figures 16A-16G, middle panels) floral tissue:

pMADSl: In V26 this gene is mainly expressed in the second and third whorls (Figure 16A, upper panel). No expression could be detected in gp (PLV), either at an early or late stage of flower development (Figure 16A, middle panel).

pMADS2: Figure 16B (upper panel) shows that the expression of this gene in V26 is mainly in the second and third whorls. In the second whorl tissue of gp (PLV) this gene is expressed at a very low level and only at young stages of flower development (Figure 16B, middle panel) whereas its expression in the third whorl (stamens) is increased in both young and mature gp (PLV) floral buds compared to that in V26.

fbpl: In V26 the fbpl gene, like pMADS2, is expressed in the second and third whorls (Figure 16C). In gp (PLV) fbpl expression is only detected in the second whorl at a very low level in early stages of development whereas no expression is detected at the late stage of second whorl development. However, in the third whorl of gp (PLV) fbpl expression is elevated both in young and mature flower buds compared to that in V26 (Figure 16C, middle panel).

pMADS3: Figure 16D shows that this gene is expressed in the third and fourth whorls of V26 (Figure 16D, upper panel) as well as in gp (PLV) (Figure 16D, middle panel). In the fourth whorl of gp (PLV) the mRNA level is slightly higher than in the wild-type V26.

pMADS4: The expression of this gene is mainly detected in the first, second and fourth whorls of V26 flowers (Figure 16E, upper panel). Expression in the mature second whorl is lower and in the mature fourth whorl is higher in gp (PLV), compared to V26 (Figure 16E, compare upper and middle panel). CHS: CHS expression is detected in all four whorls of the V26 flower, but the expression is elevated in the mature petal tissue. In the mutant gp (PLV) the CHS gene is also expressed in all four whorls (Figure 16F, middle panel); however, its up-regulation in the mature second whorl is no longer detected. By contrast, in the third whorl of the gp (PLV) flowers the CHS expression level is higher compared to that of mature stamens of V26.

CAB: The CAB gene expression is high in the first two whorl of both V26 (Figure 16G, upper panel) and gp (PLV) (Figure 16G, middle panel). In the mature wild type flower the CAB gene expression level diminishes in petals and carpels and the mRNA is not detected in mature stamens.

pMADSl is deleted from the genome of gp (PLV).

The lack of any pMADSl expression in gp (PLV) prompted us to analyze its genomic DNA for the state of the pMADSl gene. Figure 17 shows the hybridization profile of a pMADSl probe to genomic DNA of three wild-type hybrid lines (V26, V30 and W115) and a segregating population of gp mutant and wild-type plants (four out of twenty plants analyzed are shown). It was found that the DNA isolated from gp plants did not hybridize to the pMADSl probe, demonstrating that this gene is deleted from the genome. Therefore, gp (PLV) is a null mutant for pMADSl. This is not supprising since the mutation in gp (PLV) was induced by gamma radiation treatment which is known to cause chromosomal deletions. Under low stringency conditions no other bands were found to be missing on the Southern blot from the gp population, indicating that no other pMADSl related gene is deleted from the gp genome. The other four petunia MADS-genes are present and intact in the mutant genome, as confirmed by Southern and Northern blot analyses (see above). The gp (PLV) phenotype is a phenotypic marker for chromosome IV of P.hybrida, thus placing the pMADSl gene on chromosome IV (de Vlaming et al., 1984). The 'green petal' phenotype has also been obtained by EMS treatment of petunia seeds (line RlOO). Southern analysis of this mutant did not reveal any difference between mutant and wild-type pMADSl restriction fragments (not shown).

pMADSl restores petal development in gp (PLV).

To prove that pMADSl is an essential gene for petunia petal development it was necessary to show that the gp phenotype can be complemented by the pMADSl gene function. Because of regeneration problems associated with gp (PLV) a cross between this mutant and V26 was performed. A plant (GP/gp) from the progeny was used for leaf disc transformation to introduce a 35S-pMADSl gene (J84, see Methods). One of the resulting transgenic plants, carrying three independentl inserts of J84 and showing an over-expression phenotype (Halfter et al., 1993; Figure 7A), was back-crossed to gp/gp (PLV) plants. The progeny plants were analyzed for the presence of the wild-type pMADSl gene and the 35S-pMADSl transgene by Southern blot hybridization and for their floral phenotype. Among the 33 progeny plants analyzed three plants were identified that neither contained a wild-type pMADSl gene nor a 35S-pMADSl transgene in their genome and these plants had a gp phenotype. Ten other plants did not contain any wild type pMADSl gene but had one or more copies of the 35S-pMADSl transgene. Four of these still exhibit the gp phenotype, suggesting a lack of complementation (Mia). One plants showed small red sectors on a sepaloid second whorl (Mlb, Figure 18B), indicating a partial complementation by the 35S-pMADSl transgene. At a later stage this same plant showed a very weak complementation (Figure 18C and D, before and after anthesis respectively). Figure 18G and 18H show that the late differentiation into petal tissue in the second whorl tissue of Mlb resulted in the presence of trichomes on the upper epidermal layer of the petal limb. Three plants showed nearly complete (e.g. Mlc, Figure 18E) and two showed complete (e.g. Mid, Figure 18F) petal development in the second whorl. In these plants the maturation rate of the petal was clearly slower compared to that of V26 (Figure 18J and 18K). The late complementation resulted in trichomes being present on the adaxial face of the petal tube in Mlc (compare inner face of wild-type tube, Figure 18L, with that of Mlc tube, Figure 18M). Figure 181 shows that the cells that constitute the second whorl sepal structure in the gp flowers can still be recognized in flowers of plants that showed partial complementation (Mlc). In plants which showed no or weak complementation of the pMADSl gene function in the second whorl, the ectopic expression of the 35S-pMADSl gene in the third whorl did not suppress the development of extra organs. However, these extra organs in the third whorl and structures on the stamen, which in gp (PLV) showed only partial petal characteristics, now developed into full petaloid tissue (Figure 18N). Unlike the filaments of the gp (PLV) flowers, the base of the filaments of the Mlc and Mid flowers were fused to the second whorl tissue like in V26.

Figure 19A shows a Northern blot analysis of RNA isolated from mature flowers of Mia plants, and Mlc plus Mid plants. In both sets of plants the pMADSl transgene shows high expression in the first two whorls and low expression in the inner two whorls. The expression of the transgene in Mia is very low (expression in third and fourth whorl is only visible after prolonged exposure), which correlates with the lack of complementation in petal development in these transgenic plants. In the Mlc and Mid plants, the restoration of petal development correlated with a high expression level of the 35S-pMADSl transgene, as well as a up-regulation of pMADS2 and fbpl expression in the second whorl, indicating the expression of these two genes in this whorl is controlled by pMADSl. Overexpression in petunia gp (PLV) of a similar pMADS2 gene construct (see Material and Methods) did not result in any restoration of petal development, nor did it affect expression of fbpl (Figure 19B)

Phenocopy of gp (PLV) by co-suppression of pMADSl.

Additional evidence for the function of pMADSl in flower development was obtained from V26 transgenic plants in which introduction of a m35S-pMADSl chimeric gene resulted in a co-suppression of pMADSl expression, in some cases leading to a complete phenocopy of gp. The co-suppression of pMADSl was manifested in a gradation of phenotypes ranging from a decrease in petal pigmentation (five out of twenty transgenic plants, SD15 see Figure 20A), reduced petal growth (one out of twenty, SD6 see Figure 20B), reduced growth and differentiation (one of twenty, SD12 see Figure 20C) to a complete lack of petal differentiation, resulting in sepaloid structures in the second whorl (one of twenty, SD3 see Figure 20D). A partial petal differentiation of the second floral whorl in SD3 plants could occur upon aging of the plant (mainly after anthesis has occurred), resulting in slightly pigmented sepaloid second whorl structures (SD3, Figure 20E). In all of the above-mentioned transgenic plants the co-suppression resulted in the formation of third whorl stamens with filaments that were not fused to the second whorl (e.g. see SD12, Figure 20F). Also, in the case of a strong co-supression phenotype the third whorl organ number was often altered by the appearance of small additional sepaloid structures between the stamen filaments (indicated by arrow, Figure 20F). The partial petal differentiation in SD3, 6 and 12 in the second whorl differed by cell layer and could occur in sharply defined sectors (SD3, Figure 20G). Figures 20H, I and J show the different end-stages of petal development that can be detected in these second whorl floral organs. In the epidermal peels the petaloid cells could be seen next to non-pigmented sepaloid cells indicating that petal differentiation is cell autonomous (Figure 201 and J). On the plants with mild to strong co-supression phenotype the filaments of the stamen showed regions with petaloid cells, pigmented with anthocyanins and occasionally a trichome (Figure 20K).

The changed phenotype of the transgenic flowers is attributed to a co-suppression of the pMADSl gene because the pMADSl mRNA steady state level was substantially reduced in these transgenic lines (see below). The co-suppression phenotype was stably inherited to the next generation for lines SD12 and SD3. The progeny from the selfed transgenic line SD15 showed a segregating population of plants among which petal development varied from wild-type (SD15a), medium petal development (SD15b) to sepaloid petals (SD15c). This was due to the segregation of three independent inserts of the m35S-pMADSl transgene (Southern blot analysis not shown). Three out of twenty progeny plants showed a floral phenotype which was not consistent with just a suppression of the pMADSl gene function. In these flowers the second whorl petals often did not develop or were fused to the third whorl stamens to form petaloid stamen (e.g. SD15d, see Figures 20L and 20M).

RNA analysis of two co-suppression plants

To demonstrate that the phenotype in the transgenic lines carrying the m35S-pMADSl transgene was due to a co-suppression of pMADSl expression, the steady state mRNA levels of the different pMADS genes during floral development in SD15c were analyzed. SD15c is a transgenic line which showed a 'green petal' phenotype (Figures 16A-G, lower panels) and in SD15d, a transgenic line which showed a limited second whorl development and petaloid stamens (Figure 16H). Because there was no clear separation between second and third whorl in SD15d, tissue of these two whorls was combined for RNA analysis. In SD15c the pMADSl mRNA steady state levels were much reduced both in the second as well as in the third whorl, indicating a co-suppression of the pMADSl trans- and endogenous gene (Figure 16A, compare lane 2 and 3 of upper and lower panel). The transgenic line SD15d had a much reduced pMADSl steady state mRNA level in young floral buds, but the pMADSl gene was not suppressed in later stages of floral development (Figure 16G, upper panel). The lack of a significant high level of pMADSl mRNA steady state level in SD15c indicates that the change in flower phenotype in these plants was indeed caused by a co-suppression of the pMADSl trans- and endogenous gene.

To see whether the co-suppression effect was specific for the pMADSl gene, the expression of pMADS2, 3, 4 and fbpl was also analyzed. In young sepaloid tissue of the second whorl of SD15c the pMADS2 and fbpl steady state mRNA level was very low, but similar to that of V26. Upon maturation of this tissue expression of both genes remained low, in sharp contrast with the increase in pMADS2 and fbpl steady state mRNA level in mature petals of untransformed V26 flowers (Figures 16B and C, compare lane 2 of upper and lower panel). In line SD15d, pMADS2 and fbpl expression in the second/third whorl increased upon maturation (Figure 16H).

RNA analysis of V26 and gp (PLV) and the pMADSl complementation plants indicated that both pMADS2 and fbpl are regulated by pMADSl (see above). Therefore, the lack of pMADS2 and fbpl expression in the second whorl of SD15c is likely due to the reduced pMADSl gene expression in this whorl, rather than a non-specific co-suppression effect of the 35S-pMADSl transgene. In the mature third floral whorl of gp (PLV) the expression of pMADS2 and fbpl is elevated (Figure 16B and 16C, middle panels). However, Northern analysis of SD15c showed that the pMADS2 gene expression is very low in the mature third whorl organs. This indicates that pMADS2 may also be a target for co-suppression by the pMADSl transgene. Also, the phenotype of plant SD15d is stronger than that of gp (PLV), a pMADSl null-mutant, suggesting that more genes than just pMADSl are being suppressed at an early stage of flower development in SD15d. The co-suppression effect has little or no influence on pMADS3 and pMADS4 expression levels. (Figure 16D and 16E, lower panel and Figure 16H).

Analyses of petunia petal development. With the co-suppression of pMADSl function and the complementation of gp

(PLV), a series of (mature) flowers with different stages of petal development was created. These flowers not only help to genetically define the function of pMADSl, but are also of use to analyze the actual process of petal growth and differentiation in petunia. Figure 21 A shows the different end-stages of second whorl organ development, starting with the gp sepal (Figure 21A-1), V26 partial co-suppression (Figure 21A-2 and -3), gp partial restoration, and ending with wild type petal. Similar end-stages of petal development are shown schematically in FigurelOB. The petal differentiation in the epidermal cell layer suppresses trichome and stomata formation, and promotes longitudinal and lateral cell divisions in the tube and the limb. The fully differentiated petaloid epidermal cell is a small, round and cone-shaped cell with a high level of pigmentation by anthocyanins. The parenchyma cells of the inner cell layers of mature petal tissue are smaller than those in mature sepal tissue, and do not show any green pigmentation. Since the (macro) surface area of mature petal tissue is approximately twice that of sepal tissue while the parenchyma cells and epidermal cells of the petal are up to five-fold smaller than those of the sepal, petal development consists of many additional cell divisions, besides the cell divisions that are necessary to make up the (default) sepal structure. The transgenic line SD15b which shows a sepaloid second whorl in the mature flower, can still develop petal-like tissue (SD15b before anthesis, Figure 17D, as well as after anthesis, Figure 17E). The same can be seen in the complementation experiment (petal sector in Mlb, Figure 20B and petal development in Mlc, Figure 20C and 20D). How far a cell can differentiate into a complete petaloid cell depends on when the genes of the petal differentiation pathway are activated. A mature sepaloid cell may not easily undergo a change in shape but become pigmented with anthocyanins, whereas a young sepaloid cell may become altered in cell shape and/or divide to give rise to a fully differentiated petaloid cell.

The growth patterns that transform a sepaloid organ into a petal are illustrated in Figure 21C. The sepal growth (S in Figure 21C) includes a congenital fusion at the base of the five sepaloid organs, leading to a tube-structure. This tube-structure corresponds to the fused part of the corolla limb; the corolla tube has no real equivalent in gp (see below). When the petal differentiation pathway is activated S-growth is transformed into C-growth (Figure 21C) and extended by additional lateral cell divisions (Cl in Figure 21C), additional longitudinal cell divisions at the base which make up a part of the corolla tube (C2 in Figure 21C) and additional cell divisions under the base of the sepal and the stamen (C3 and F2 in Figure 21C) which make up the part of the corolla tube with the fused stamen filaments. The C3 and F2 growth are most easily affected by pMADSl co-suppression, and least easily complemented by pMADSl expression in gp (PLV) plants. Under conditions of partial co-suppression (SD15a) or partial - complementation (Mlb) of the pMADSl function, the tube and filament growth only occurs above the petal and stamen initiation zone (C2 and FI, Figure 21C), resulting in separate, (non-fused) stamen filaments and corolla tube. The differentiation of the sepaloid second whorl organ into petal is mainly responsible for the corolla limb structure (C and Cl in Figure 21C). This is for instance illustrated by flowers of Mlb (Figure 18D, G and H) which show petal tissue (Cl growth) at the fringes of the otherwise sepaloid second whorl organs and in flowers of Mlc (Figure 181), where the sepaloid structure that forms in gp (PLV) flowers can still be seen within the corolla limb that has formed by pMADSl complementation. The corolla tube is formed by C2 and C3 growth (Figure 21C), and equivalent growth is either extremely limited or absent in the sepaloid organs of gp.

Discussion

The functions of pMADSl in determining floral organ identity.

Evidence is presented that the mutant gp (PLV) suffers a chromosomal deletion that includes the pMADSl locus. Moreover, petal development in gp can be restored by a 35S-pMADSl transgene, while the gp phenotype is obtained by pMADSl co-suppression. These results combined indicate that the pMADSl -gene can be designated as GP. Although pMADSl does not control stamen growth in the third whorl, it does have minor effects in this whorl by suppressing formation of petaloid cells and additional organs. The petal differentiation pathway appears to be dosage dependent, since the degree of complementation is correlated with the expression level Figure 19A) and copy number of the 35S-pMADSl transgene (Figure 18E and F). Partial complementation by the 35S-pMADSl transgene could occur in defined sectors (Figure 16B). Such a sector is phenotypically similar to a sector in the DEFA (def-621) mutant in which somatic reversion has occured during second whorl development, resulting in the restoration of DEFA expression (Carpenter and Coen, 1990). However, the sector in Mlb is genetically different because in this tissue the pMADSl gene is under control of the 'constitutive' CaMV 35S-promoter. This result suggests that a weakly expressed pMADSl transgene can activate petal differentiation in a cell at a low frequency, while subsequently feedback mechanisms might re-enforce this differentiation process. In this connection it should also be noted that a putative MADS-box DNA binding-site (CCAAAGATGG) is present in the CaMV 35S-promoter. Therefore, expression from this promoter might also be subject to regulation by MADS-box genes.

pMADSl regulates the expression of pMADS2 and fbpl in the second whorl. Since pMADS-box gene expression varies throughout floral development and at similar stages among different Petunia hybrida lines, caution should be exercised in interpretating differences in expression levels among plants with different genetic backgrounds. The effect of the absence of pMADSl on pMADS2 and fbpl expression, however, was consistently observed (Figures 16B and C, Figure 19A). In the second whorl pMADSl up-regulates, whereas in the third whorl it down-regulates pMADS2 and fbpl expression (compare expression in V26 and gp (PLV), Figures 16A, B and C). pMADSl has also been shown to up-regulate its own and pMADS2 expression in first whorl tissue of transgenic plants in which the ectopic expression of pMADSl resulted in the homeotic conversion of sepals to petals (Halfter et al., 1993). The expression of fbpl in another 'green petal' mutant (M68) has been reported previously (Angenent et al, 1992) but the pMADSl expression in this line has not been described. Also, in these analyses the effect of floral development on the expression levels of fbpl was not considered. The expression of pMADS3 was largely unaffected by the presence or absence of pMADSl gene expression, as was pMADS4 gene expression.

The observation that petaloid cells can develop in a pMADSl null mutant (Figure 15H and 15J) suggests that pMADSl controls petal cell differentiation indirectly. For example, it might regulate the expression of regulatory factors in the second whorl that directly interact with petal specific genes. These 'downstream' transcription factors apparently do not need the pMADSl gene for their expression in cells of the third whorl. It is noted that the expression profile of both pMADS2 and fbpl fit that of the above described 'petal-specific' downstream genes.

Co-suppression of pMADSl.

The phenomenon of co-suppression was first described by Napoli et al. (1990) and van der Krol et al. (1990) and occurs when a transgene somehow represses its own expression as well as that of other homologous genes, either wild-type genes or other transgenes, present in the plant genome, (Napoli et al., 1990). Although co-suppression has now been observed with many different types of genes in transgenic plants, its molecular mechanism remains unknown (reviewed by Jorgensen, 1990). A co-suppression phenotype was observed with pMADSl but not with a simmilar pMADS2 gene construct (van der Krol, unpublished results). In the pMADSl co-suppression plants expression of the transgene was not detected in sepals of line SD15c and d (Figure 16A, lower panel and Figure 16H, upper panel), indicating that in both these lines the pMADSl transgene(s) is already in a suppressed state. This has also been observed for other cases of co-suppression (e.g. chalcone synthase and the dihydro-flavonol reductase genes, van der Krol et al., 1990). Most pMADSl co-suppression plants showed a phenotype varying from wild-type to that of gp, indicating that the changes can indeed be ascribed to alterations in pMADSl expression level. However, there is some evidence suggesting that the co-suppression may not always be specific for pMADSl. The change in fbpl expression in the co-suppression plant SD15c is comparible to the change in fbpl expression in gp, however the changes in pMADS2 expression do not. It could be that pMADS2 is also a target for the co-suppression in this transgenic line. Moreover, the SD15d flowers show a phenotype more severe than gp flowers, indicating co-suppression of gene functions other than those of pMADSl.

Differences between petunia and Antirrhinum and Arabidopsis flower development. The deduced function of pMADSl in second whorl organ development agrees with that of DEFA and AP3. Mutation of one of these genes produces a homeotic conversion of petal into sepal. Both pMADS2 and fbpl show a very low level of expression at early stages of second whorl organ development, and in the absence of pMADSl this expression fails to increase to the high level detected in the wild-type mature petal. This expression pattern is similar to what has been described for DEFA and GLO in Antirrhinum. (Sommer et al, 1990 and Trobner et al., 1992) and AP3 and Pi in Arabidopsis (Jack et al., 1990). Both set of B-type genes are independently induced at early stages of flower development but show an inter-dependency for maintenance and increase of expression at later stages of petal development. Sommer (1990) and Trobner (1992) reported that only the DEF/GLO heterodimer can bind to DNA target sites located in the promoter rgions of DEFA and GLO. This result suggests that both genes are involved in up-regulating their own expression (Trobner et al., 1992).

In contrast to DEFA and AP3, the petunia pMADSl is genetically redundant for third whorl stamen development. A minor effect of pMADSl in the third whorl is the suppression of petaloid cell formation on the stamen filaments. A similar third-whorl function would be masked in DEFA and AP3 mutants because they do not develop stamens. In petunia stamen development in the absence of pMADSl coincides with the up-regulation of pMADS2 and fbpl expression. This is in sharp contrast to the situation in Antirrhinum and Arabidopsis where, as in the second whorl, both set of B-type genes show an inter-dependency for maintenance of expression in the third whorl organs (Trobner et al., 1992; Jack et al., 1992). Thus, pMADS2 and fbpl proteins can function without the pMADSl protein, indicating a different type of interaction between the petunia proteins pMADSl and pMADS2/fbpl on one hand, and between the Antirrhinum proteins DEFA and GLO and the Arabidopsis proteins AP3 and Pi on the other hand.

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This invention may be embodied in other forms or carried out in other ways without departing from the spirit or essential characteristics thereof. The present disclosure is therefore to be considered as in all respects illustrative and not restrictive, the scope of the invention being indicated by the appended Claims, and all changes which come within the meaning and range of equivalency are intended to be embraced therein.

Claims

WHAT IS CLAIMED IS:

1. A method for controlling the morphogenesis of at least one plant organ comprising modulating the expression of at least one of the homeotic genes that determines the identity of said organ.

2. The method of Claim 1 wherein the said at least one homeotic gene is overexpressed.

3. The method of Claim 1 wherein the said at least one homeotic gene is effectively underexpressed.

4. The method of Claim 2 wherein the plant organ comprises the flower, and the method is applied to modify the number of the petals presented.

5. The method of Claim 3 wherein the plant organ comprises the flower, and the method is applied to modify the structure of the petals presented.

6. The method of Claim 4 wherein the plant is petunia, and an additional whorl of petals is presented.

7. The method of Claim 5 wherein the plant is petunia, and the petals are converted to present at least a partial sepaloid structure.

8. The method of Claim 2 including isolating the homeotic gene to be overexpressed.

9. The method of Claim 3 including isolating the homeotic gene to be underexpressed.

10. The method of Claim 1 wherein the homeotic gene comprises the flower gene green petal in petunia.

11. The method of Claim 2 wherein said homeotic gene is overexpressed by ectopic expression.

12. The method of Claim 11 wherein said ectopic expression comprises introducing said homeotic gene into the plant transformation vector pMON 530, and transferring said vector to Agrobacterium tumefaciens strain GV31111SE.

13. A DNA sequence or degenerate variant thereof, which encodes the homeotic flower gene green petal in petunia, or a fragment thereof, selected from the group consisting of: (A) the DNA sequence of FIGURE 2; (B) DNA sequences that hybridize to the DNA sequence of FIGURE 2 under standard hybridization conditions; and (C) DNA sequences that code on expression for an amino acid sequence encoded by the foregoing DNA sequence of FIGURE 2.

14. A recombinant DNA molecule comprising a DNA sequence or degenerate variant thereof, which encodes the homeotic flower gene green petal in petunia, or a fragment thereof, selected from the group consisting of: (A) the DNA sequence of FIGURE 2; (B) DNA sequences that hybridize to the foregoing DNA sequence under standard hybridization conditions; and (E) DNA sequences that code on expression for an amino acid sequence encoded by the foregoing DNA sequence.

15. The DNA sequence of either of Claims 13 or 14, operatively linked to an expression control sequence.

16. The DNA sequence of Claim 15, wherein said expression control sequence is selected from the group consisting of the CaMV 19S promoter, the CaMV 35S promoter, the rbcs E9 gene from pea, the wheat histone H3 gene, the nopaline synthase gene of the Ti plasmid of Agrobacterium tumefaciens, the octopine synthase gene of the Ti plasmid of Agrobacterium tumefaciens, the mannopine synthase promoter, the light-inducible promoter from the small subunit of ribulose bis-phosphate carboxylase (ssRUBISCO), and combinations thereof.

17. A plant transformation vector containing a DNA sequence or degenerate variant thereof, which encodes the homeotic gene green petal in petunia, or a fragment thereof, selected from the group consisting of: (A) the DNA sequence of FIGURE 2; (B) DNA sequences that hybridize to the foregoing DNA sequence under standard hybridization conditions; and (C) DNA sequences that code on expression for an amino acid sequence encoded by the foregoing DNA sequence.

18. The plant transformation vector of Claim 17 comprising the vector pMON 530.

19. The method of Claim 3 wherein said homeotic gene is underexpressed by the introduction of a transgene corresponding to said homeotic gene.

20. The method of Claim 19 wherein said introduction comprises introducing DNA corresponding to said homeotic gene into the plant transformation vector pMON721, and transferring said vector to Agrobacterium tumefaciens.

21. A promoter for combination with the DNA sequence or degenerate variant thereof, which encodes the homeotic flower gene green petal in petunia, or a fragment thereof, comprising a modified CaMV 35S promoter (m35S), said m35S comprising the construct VIP149 depicted in FIGURE 5A.

22. The promoter of Claim 21 comprised of the -90 to +8 fragment of the CaMV 35S promoter and a fragment corresponding to four ligated fragments comprised of two alternations of each of the DNA sequences denominated 4xB3 and 2xTX4, respectively, set forth in FIGURE 5B and in respective Sequence Identification No. 2 (SEQ ID NO:2) and Sequence Identification No. 3 (SEQ ID NO:3).

23. A recombinant DNA molecule comprising a DNA sequence or degenerate variant thereof, which encodes the homeotic flower gene green petal in petunia, or a fragment thereof, operatively linked to an expression control sequence, said expression control sequence comprising a modified CaMV 35S promoter (m35S), said m35S comprising the construct VIP149 depicted in FIGURE 5A.

24. A petunia flower having an additional whorl of petals prepared by the method of Claim 2.

25. A petunia flower having two whorls of petals, and functional carpel and stamen structures.

26. The petunia flower of Claim 25 wherein the whorl of petals furthest removed from said carpel and said stamen structures is derived from sepals.

27. A petunia flower having two whorls of petals, and functional carpel and stamen structures.

28. A petunia flower having a whorl of petals defining sepaloid characteristics along at least a portion thereof prepared by the method of Claim 3.

29. A petunia flower having a whorl of petals defining sepaloid characteristics along at least a portion thereof.

30. The petunia flower of Claim 29 selected from the flowers depicted in FIGURES 10A-10D.