Co-suppression of the petunia homeotic gene fbp2 affects the identity of the generative meristem

The function of the petunia MADS box gene fbp2 in the control of floral development has been investigated. Inhibition of fbp2 expression in transgenic plants by a co-suppression approach resulted in the development of highly aberrant flowers with modified whorl two, three and four organs. This mutant flower phenotype inherited as a single Mendelian trait. The flowers possess a green corolla which is reduced in size. Furthermore, the stamens are replaced by green petaloid structures and the inner gynoecial whorl is dramatically reduced. No ovules or placenta are formed and instead two new inflorescences developed in the axils of the carpels. These homeotic transformations are accompanied by a complete down-regulation of the petunia MADS box gene fbp6 which is highly homologous to the Arabidopsis and Antirrhinum genes agamous (ag) and plena (ple). In contrast to this, two other petunia MADS box genes, exclusively expressed in whorls two and three, are still transcribed. Our results indicate that the fbp2 gene belongs to a new class of morphogenesis genes involved in the determination of the central part of the generative meristem.     

Isolation of the tomato AGAMOUS gene TAG1 and analysis of its homeotic role in transgenic plants.

To understand the details of the homeotic systems that govern flower development in tomato and to establish the ground rules for the judicious manipulation of this floral system, we have isolated the tomato AGAMOUS gene, designated TAG1, and examined its developmental role in antisense and sense transgenic plants. The AGAMOUS gene of Arabidopsis is necessary for the proper development of stamens and carpels and the prevention of indeterminate growth of the floral meristem. Early in flower development, TAG1 RNA accumulates uniformly in the cells fated to differentiate into stamens and carpels and later becomes restricted to specific cell types within these organs. Transgenic plants that express TAG1 antisense RNA display homeotic conversion of third whorl stamens into petaloid organs and the replacement of fourth whorl carpels with pseudocarpels bearing indeterminate floral meristems with nested perianth flowers. A complementary phenotype was observed in transgenic plants expressing the TAG1 sense RNA in that first whorl sepals were converted into mature pericarpic leaves and sterile stamens replaced the second whorl petals.     

Negative regulation of the Arabidopsis homeotic gene AGAMOUS by the APETALA2 product.

We characterized the distribution of AGAMOUS (AG) RNA during early flower development in Arabidopsis. Mutations in this homeotic gene cause the transformation of stamens to petals in floral whorl 3 and of carpels to another ag flower in floral whorl 4. We found that AG RNA is present in the stamen and carpel primordia but is undetectable in sepal and petal primordia throughout early wild-type flower development, consistent with the mutant phenotype. We also analyzed the distribution of AG RNA in apetela2 (ap2) mutant flowers. AP2 is a floral homeotic gene that is necessary for the normal development of sepals and petals in floral whorls 1 and 2. In ap2 mutant flowers, AG RNA is present in the organ primordia of all floral whorls. These observations show that the expression patterns of the Arabidopsis floral homeotic genes are in part established by regulatory interactions between these genes.     

The TM5 MADS Box Gene Mediates Organ Differentiation in the Three Inner Whorls of Tomato Flowers

The tomato MADS box gene no. 5 (TM5) is shown here to be expressed in meristematic domains fated to form the three inner whorls-petals, stamens, and gynoecia-of the tomato flower. TM5 is also expressed during organogenesis and in the respective mature organs of these three whorls. This is unlike the major organ identity genes of the MADS box family from Antirrhinum and Arabidopsis, which function in overlapping primordial territories consisting of only two floral whorls each. The developmental relevance of the unique expression pattern of this putative homeotic gene was examined in transgenic plants. In agreement with the expression patterns, antisense RNA of the TM5 gene conferred both early and late alterations of morphogenetic markers. Early defects consist of additional whorls or of a wrong number of organs per whorl. Late, organ-specific changes include evergreen, cauline, and unabscised petals; green, dialytic, and sterile anthers; and sterile carpels and defective styles on which glandular trichomes characteristic of sepals and petals are ectopically formed. However, a complete homeotic transformation of either organ was not observed. The early and late floral phenotypes of TM5 antisense plants suggest that TM5 mediates two unrelated secondary regulatory systems. One system is the early function of the floral meristem identity genes, and the other system is the function of the major floral organ identity genes.     

Toward the analysis of the petunia MADS box gene family by reverse and forward transposon insertion mutagenesis approaches: B, C, and D floral organ identity functions require SEPALLATA-like MADS box genes in petunia

We have initiated a systematic functional analysis of the MADS box, intervening region, K domain, C domain-type MADS box gene family in petunia. The starting point for this has been a reverse-genetics approach, aiming to select for transposon insertions into any MADS box gene. We have developed and applied a family signature insertion screening protocol that is highly suited for this purpose, resulting in the isolation of 32 insertion mutants in 20 different MADS box genes. In addition, we identified three more MADS box gene insertion mutants using a candidate-gene approach. The defined insertion lines provide a sound foundation for a systematic functional analysis of the MADS box gene family in petunia. Here, we focus on the analysis of Floral Binding Protein2 (FBP2) and FBP5 genes that encode the E-function, which in Arabidopsis has been shown to be required for B and C floral organ identity functions. fbp2 mutants display sepaloid petals and ectopic inflorescences originating from the third floral whorl, whereas fbp5 mutants appear as wild type. In fbp2 fbp5 double mutants, reversion of floral organs to leaf-like organs is increased further. Strikingly, ovules are replaced by leaf-like structures in the carpel, indicating that in addition to the B- and C-functions, the D-function, which specifies ovule development, requires E-function activity. Finally, we compare our data with results obtained using cosuppression approaches and conclude that the latter might be less suited for assigning functions to individual members of the MADS box gene family.     

Multiple AGAMOUS homologs from cucumber and petunia differ in their ability to induce reproductive organ fate.

The C function in Arabidopsis, which specifies stamen and carpel identity, is represented by a single gene called AGAMOUS (AG). From both petunia and cucumber, two MADS box genes have been isolated. Both share a high degree of amino acid sequence identity with the Arabidopsis AG protein. Their roles in specifying stamen and carpel identity have been studied by ectopic expression in petunia, resulting in plants with different floral phenotypes. Cucumber MADS box gene 1 (CUM1) induced severe homeotic transformations of sepals into carpelloid structures and petals into stamens, which is similar to ectopic AG expression in Arabidopsis plants. Overexpression of the other cucumber AG homolog, CUM10, resulted in plants with partial transformations of the petals into antheroid structures, indicating that CUM10 is also able to promote floral organ identity. From the two petunia AG homologs pMADS3 and Floral Binding Protein gene 6 (FBP6), only pMADS3 was able to induce homeotic transformations of sepals and petals. Ectopic expression of both pMADS3 and FBP6, as occurrs in the petunia homeotic mutant blind, phenocopies the pMADS3 single overexpresser plants, indicating that there is no additive effect of concerted expression. This study demonstrates that in petunia and cucumber, multiple AG homologs exist, although they differ in their ability to induce reproductive organ fate.     

Stamenless, a tomato mutant with homeotic conversions in petals and stamens

A tomato (Lycopersicon esculentum Mill.) monogenic semidominant mutation, stamenless (sl), which results in homeotic conversions in two adjacent floral whorls, was studied. When grown at standard temperature, flowers of sl/sl plants showed sepaloid petals in the second whorl and strong transformation of stamens to carpels in whorl three. These transformed carpels were fused with each other and with the genuine carpels in the fourth whorl to form a unique gynoecium. The mutation is semidominant since heterozygous plants showed a phenotype intermediate between that of the wild type (WT) and that of homozygous mutant plants, with nearly WT petals but with feminized stamens bearing naked ovules on the base of their adaxial face. The initiation and position of organ primordia in sl/sl flowers were not altered when compared with WT primordia although development of organ primordia in the second and third whorls deviated from WT at an early stage as observed by scanning electron microscopy. The mutant phenotype is temperature sensitive and when sl/sl plants were cultured at low temperature, the morphology of some flowers resembled that of the WT. This reversion of the mutant phenotype is also induced by treatment of young sl/sl plants with gibberellic acid, providing evidence that gibberellin synthesis or sensitivity could mediate the effect of low temperature on the mutant phenotype. Southern blot analyses using a Deficiens-homologous gene from Solanum tuberosum as a probe showed a restriction-fragment-length polymorphism (RFLP) linked to the sl mutation. This result indicates that the mutation affects a Deficiens-like gene that controls the identity of petals and stamens. Received: 10 December 1998 / Accepted: 29 March 1999     

Role of petunia pMADS3 in determination of floral organ and meristem identity,as revealed by its loss of function

pMADS3, a petunia class C gene, is the candidate homologue of Arabidopsis AGAMOUS (AG), which is involved in the specification of stamens and carpels. We report the characterization of loss-of-function phenotype of pMADS3 that resulted from silencing of this gene. Silencing of pMADS3 resulted in homeotic conversion of stamens into petaloid structures, whereas the carpels were only weakly affected. Ectopic secondary inflorescences emerged from the interstamenal region in the third whorl, which is unique and has not been reported for any class C gene of other plant species. Third-order inflorescences emerged at corresponding positions in the third whorl of inner flowers of secondary inflorescences, indicating reiterative conversion of parts of the floral meristem into inflorescence meristem. On the basis of phenotypic analysis of the pMADS3-silenced plants, we propose that pMADS3 is involved in determination of floral organ and floral meristem identity in petunia. Two hybrid studies in yeast showed that PMADS3 protein interacted specifically with FBP2, a candidate homologue of Arabidopsis SEPALLATA3 (SEP3). The evidence presented here suggest that a complex involving PMADS3 and FBP2 is responsible for specification of organ identity in the third whorl.     

Genetic separation of third and fourth whorl functions of AGAMOUS.

AGAMOUS (AG) is an Arabidopsis MADS box gene required for normal development of the third and fourth whorls of the flower. In previously described ag mutants, the third whorl stamens are replaced by petals, and the fourth whorl is replaced by another (mutant) flower. We describe two new ag alleles, ag-4 and AG-Met205, retaining partial AG activity. Both produce flowers with stamens in the third whorl and indeterminate floral meristems; however, ag-4 flowers contain sepals in the fourth whorl, and AG-Met205 produces carpels. The ag-4 mutation results in partial loss of the C terminus of the K domain, a putative coiled coil, and AG-Met205 contains a site-directed mutation that causes a single amino acid change in this same region of the K box. Two models that might explain how these changes in AG result in the separation of different AG activities are discussed.     

Expression of the Arabidopsis floral homeotic gene AGAMOUS is restricted to specific cell types late in flower development.

Mutations in the AGAMOUS (AG) gene cause transformations in two adjacent whorls of the Arabidopsis flower. Petals develop in the third floral whorl rather than the normal stamens, and the cells that would normally develop into the fourth whorl gynoecium behave as if they constituted an ag flower primordium. Early in flower development, AG RNA is evenly distributed throughout third and fourth whorl organ primordia but is not present in the organ primordia of whorls one and two. In contrast to the early expression pattern, later in flower development, AG RNA is restricted to specific cell types within the stamens and carpels as cellular differentiation occurs in those organs. Ectopic AG expression patterns in flowers mutant for the floral homeotic gene APETELA2 (AP2), which regulates early AG expression, suggest that the late AG expression is not directly dependent on AP2 activity.     

Ectopic expression of pMADS3 in transgenic petunia phenocopies the petunia blind mutant.

We cloned a MADS-box gene, pMADS3, from Petunia hybrida, which shows high sequence homology to the Arabidopsis AGAMOUS and Antirrhinum PLENA. pMADS3 is expressed exclusively in stamens and carpels of wild-type petunia plants. In the petunia mutant blind, which shows homeotic conversions of corolla limbs into antheroid structures with pollen grains and small parts of sepals into carpelloid tissue, pMADS3 is expressed in all floral organs as well as in leaves. Ectopic expression of pMADS3 in transgenic petunia leads to phenocopies of the blind mutant, i.e., the formation of antheroid structures on limbs and carpelloid tissue on sepals. Transgenic tobacco plants that overexpress pMADS3 exhibit an even more severe phenotype, with the sepals forming a carpel-like structure encasing the interior floral organs. Our results identify BLIND as a negative regulator of pMADS3, which specifies stamens and carpels during petunia flower development.     

Manipulation of flower structure in transgenic tobacco.

Genetic studies suggest that three homeotic functions, designated A, B, and C, act alone and together to specify the fate of floral organ primordia in distantly related dicotyledonous plant species. To test the genetic model, we have generated transgenic tobacco plants that ectopically express the AGAMOUS gene from Brassica napus, which is necessary for the C function. Flowers on the resulting plants showed homeotic transformations of sepals into carpels and petals into stamens. These phenotypes are consistent with predictions from the genetic model, show that expression of AGAMOUS is sufficient to provide ectopic C function, and demonstrate that the structure of flowers can be manipulated in a predictable manner by altering the expression of a single regulatory gene. Furthermore, the generation of the predicted transformations by ectopic expression of the Brassica gene in transgenic tobacco indicates that gene functions are interchangeable between phylogenetically distant species.     

The duplicated B-class heterodimer model: whorl-specific effects and complex genetic interactions in Petunia hybrida flower development

In both Antirrhinum (Antirrhinum majus) and Arabidopsis (Arabidopsis thaliana), the floral B-function, which specifies petal and stamen development, is embedded in a heterodimer consisting of one DEFICIENS (DEF)/APETALA3 (AP3)-like and one GLOBOSA (GLO)/PISTILLATA (PI)-like MADS box protein. Here, we demonstrate that gene duplications in both the DEF/AP3 and GLO/PI lineages in Petunia hybrida (petunia) have led to a functional diversification of their respective members, which is reflected by partner specificity and whorl-specific functions among these proteins. Previously, it has been shown that mutations in PhDEF (formerly known as GREEN PETALS) only affect petal development. We have isolated insertion alleles for PhGLO1 (FLORAL BINDING PROTEIN1) and PhGLO2 (PETUNIA MADS BOX GENE2) and demonstrate unique and redundant properties of PhDEF, PhGLO1, and PhGLO2. Besides a full homeotic conversion of petals to sepals and of stamens to carpels as observed in phglo1 phglo2 and phdef phglo2 flowers, we found that gene dosage effects for several mutant combinations cause qualitative and quantitative changes in whorl 2 and 3 meristem fate, and we show that the PHDEF/PHGLO1 heterodimer controls the fusion of the stamen filaments with the petal tube. Nevertheless, when the activity of PhDEF, PhGLO1, and PhGLO2 are considered jointly, they basically appear to function as DEF/GLO does in Antirrhinum and to a lesser extent as AP3/PI in Arabidopsis. By contrast, our data suggest that the function of the fourth B-class MADS box member, the paleoAP3-type PETUNIA HYBRIDA TM6 (PhTM6) gene, differs significantly from the known euAP3-type DEF/AP3-like proteins; PhTM6 is mainly expressed in the developing stamens and ovary of wild-type flowers, whereas its expression level is upregulated in whorls 1 and 2 of an A-function floral mutant; PhTM6 is most likely not involved in petal development. The latter is consistent with the hypothesis that the evolutionary origin of the higher eudicot petal structure coincided with the appearance of the euAP3-type MADS box genes.     

Spatial distribution of the RABBIT EARS protein and effects of its ectopic expression in Arabidopsis thaliana flowers

In many flowering plants, flowers consist of two peripheral organs, sepals and petals, occurring in outer two whorls, and two inner reproductive organs, stamens and carpels. These organs are arranged in a concentric pattern in a floral meristem, and the organ identity is established by the combined action of floral homeotic genes expressed along the whorls. Floral organ primordia arise at fixed positions in the floral meristem within each whorl. The RABBIT EARS (RBE) gene is transcribed in the petal precursor cells and primordia, and regulates petal initiation and early growth in Arabidopsis thaliana. We investigated the spatial and temporal expression pattern of a RBE protein fused to the green fluorescent protein (GFP). Expression of the GFP:RBE fusion gene under the RBE cis-regulatory genomic fragment rescues the rbe petal defects, indicating that the fusion protein is functional. The GFP signal is located to the cells where RBE is transcribed, suggesting that RBE function is cell-autonomous. Ectopic expression of GFP:RBE under the APETALA1 promoter causes the homeotic conversion of floral organs, resulting in sterile flowers. In these plants, the class B homeotic genes APETALA3 and PISTILLATA are down-regulated, suggesting that the restriction of the RBE expression to the petal precursor cells is crucial for flower development.     

A new role of the Arabidopsis SEPALLATA3 gene revealed by its constitutive expression

During Arabidopsis flower development a set of homeotic genes plays a central role in specifying the distinct floral organs of the four whorls, sepals in the outermost whorl, and petals, stamens, and carpels in the sequentially inner whorls. The current model for the identity of the floral organs includes the SEPALLATA genes that act in combination with the A, B and C genes for the specification of sepals, petals, stamens and carpels. According to this new model, the floral organ identity proteins would form different complexes of proteins for the activation of the downstream genes. We show that the presence of SEPALLATA proteins is needed to activate the AG downstream gene SHATTERPROOF2, and that SEPALLATA4 alone does not provide with enough SEPALLATA activity for the complex to be functional. Our results suggest that CAULIFLOWER may be part of the protein complex responsible for petal development and that it is fully required in the absence of APETALA1 in 35S::SEP3 plants. In addition, genetic and molecular experiments using plants constitutively expressing SEPALLATA3 revealed a new role of SEPALLATA3 in activating other B and C function genes. We molecularly prove that the ectopic expression of SEPALLATA3 is sufficient to ectopically activate APETALA3 and AGAMOUS. Remarkably, plants that constitutively express both SEPALLATA3 and LEAFY developed ectopic petals, carpels and ovules outside of the floral context.     

Eucalyptus has a functional equivalent of the Arabidopsis floral meristem identity gene LEAFY

Two genes cloned from Eucalyptus globulus, Eucalyptus LeaFy (ELF1 and ELF2), have sequence homology to the floral meristem identity genes LEAFY from Arabidopsis and FLORICAULA from Antirrhinum. ELF1 is expressed in the developing eucalypt floral organs in a pattern similar to LEAFY while ELF2 appears to be a pseudo gene. ELF1 is expressed strongly in the early floral primordium and then successively in the primordia of sepals, petals, stamens and carpels. It is also expressed in the leaf primordia and young leaves and adult and juvenile trees.The ELF1 promoter coupled to a GUS reporter gene directs expression in transgenic Arabidopsis in a temporal and tissue-specific pattern similar to an equivalent Arabidopsis LEAFY promoter construct. Strong expression is seen in young flower buds and then later in sepals and petals. No expression was seen in rosette leaves or roots of flowering plants or in any non-flowering plants grown under long days. Furthermore, ectopic expression of the ELF1 gene in transgenic Arabidopsis causes the premature conversion of shoots into flowers, as does an equivalent 35S-LFY construct. These data suggest that ELF1 plays a similar role to LFY in flower development and that the basic mechanisms involved in flower initiation and development in Eucalyptus are similar to those in Arabidopsis.     

Functional diversification of the two C-class MADS box genes OSMADS3 and OSMADS58 in Oryza sativa

The C-class MADS box gene AGAMOUS (AG) plays crucial roles in Arabidopsis thaliana development by regulating the organ identity of stamens and carpels, the repression of A-class genes, and floral meristem determinacy. To examine the conservation and diversification of C-class gene function in monocots, we analyzed two C-class genes in rice (Oryza sativa), OSMADS3 and OSMADS58, which may have arisen by gene duplication before divergence of rice and maize (Zea mays). A knockout line of OSMADS3, in which the gene is disrupted by T-DNA insertion, shows homeotic transformation of stamens into lodicules and ectopic development of lodicules in the second whorl near the palea where lodicules do not form in the wild type but carpels develop almost normally. By contrast, RNA-silenced lines of OSMADS58 develop astonishing flowers that reiterate a set of floral organs, including lodicules, stamens, and carpel-like organs, suggesting that determinacy of the floral meristem is severely affected. These results suggest that the two C-class genes have been partially subfunctionalized during rice evolution (i.e., the functions regulated by AG have been partially partitioned into two paralogous genes, OSMADS3 and OSMADS58, which were produced by a recent gene duplication event in plant evolution).