CaMADS1, an AGAMOUS homologue from hazelnut, produces floral homeotic conversion when expressed in Arabidopsis

CaMADS1 is a floral-specific MADS box gene of hazelnut (Corylus avellana) which, according to its sequence and expression pattern, belongs to the AGAMOUS gene sub-family. To investigate whether CaMADS1 plays a role in specifying stamen and carpel identity, this gene was ectopically expressed in Arabidopsis. The constitutive expression of CaMADS1 in transgenic plants produced the homeotic conversion of first and second whorl organs: the first whorl exhibited carpelloid sepals and the second whorl showed staminoid features. This was expected on the basis of the ABC model, according to which ectopic expression of a functional AGAMOUS (a gene of class C) orthologue would suppress the A class homeotic function in the first and second whorls, leading to transformation of these whorls into carpels and stamen, respectively. These results indicate a functional equivalency between AGAMOUS and CaMADS1, for which CaMADS1 might behave like a class C homeotic gene, controlling the determination of stamen and carpel identity in hazelnut Received: 31 July 2000 / Revision accepted: 28 September 2000     

Characterization of oil palm MADS box genes in relation to the mantled flower abnormality

In vitro propagation of oil palm (Elaeis guineensis Jacq.) frequently induces a somaclonal variant called ‘mantled’ abnormality, in which the stamens of both male and female flowers are transformed into carpels. This leads to a reduced yield or complete loss of the harvest of palm oil. The high frequency of the abnormality in independent lines and the high reversal rate suggest that it is due to an epigenetic change. The type of morphological changes suggest that it involves homeotic MADS box genes that regulate the identity of the flower whorls. We have isolated a number of MADS box genes from oil palm inflorescences by a MADS box-directed mRNA display approach. The isolated partial cDNAs included genes that were likely to function at the initial stages of flowering as well as genes that may function in determination of the inflorescence and the identity of the flower whorls. For four genes that were homologous to genes known to affect the reproductive parts of the flower, full length cDNAs were isolated. These were a B-type MADS box gene which may function in the determination of stamen formation, a C-type gene expected to be involved in stamen and carpel formation, and two putative SEP genes which act in concert with the A-, B- and C-type MADS box gene in determining flower whorl formation. The B-type gene EgMADS16 was functionally characterized as a PISTILLATA orthologue; it was able to complement an Arabidopsis thaliana pi mutant. Whether EgMADS16, or any of the other EgMADS genes, are functionally involved in the mantled condition remains to be established.     

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.     

Interactions between gene activity and cell layers during floral development

The DEFICIENS (DEF) gene is required for establishing petal and stamen identity in Antirrhinum and is expressed in all three layers of the floral meristem in whorls 2 and 3. Expression of DEF in a subset of meristem layers gives rise to organs with characteristic shapes and cell types, reflecting altered patterns and levels of DEF gene activity. To determine how the contributions of layers and gene activity interact, we exploited a DEF allele which carries a transposon insertion in the MADS box region to generate periclinal chimeras expressing alleles with different activities. By comparing the phenotype, development and expression patterns of these chimeras we show that expression of DEF in L1 makes a major contribution to morphology in whorl 2, irrespective of the allele. By contrast L1 expression is largely unable to rescue whorl 3, possibly because of a non-autonomous inhibitor of DEF activity in this whorl.     

Regulation of SUP expression identifies multiple regulators involved in arabidopsis floral meristem development

During the course of flower development, floral homeotic genes are expressed in defined concentric regions of floral meristems called whorls. The SUPERMAN (SUP, also called FLO10) gene, which encodes a C2H2-type zinc finger protein, is involved in maintenance of the stamen/carpel whorl boundary (the boundary between whorl 3 and whorl 4) in Arabidopsis. Here, we show that the regulation of SUP expression in floral meristems is complex, consisting of two distinct phases, initiation and maintenance. The floral meristem identity gene LEAFY (LFY) plays a role in the initiation phase through at least two pathways, which differ from each other in the involvement of two homeotic genes, APETALA3 (AP3) and PISTILLATA (PI). AP3, PI, and another homeotic gene, AGAMOUS (AG), are further required for SUP expression in the later maintenance phase. Aside from these genes, there are other as yet unidentified genes that control both the temporal and spatial patterns of SUP expression in whorl 3 floral meristems. SUP appears to act transiently, probably functioning to trigger a genetic circuit that creates the correct position of the whorl 3/whorl 4 boundary.     

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.     

The seirena B Class Floral Homeotic Mutant of California Poppy (Eschscholzia californica) Reveals a Function of the Enigmatic PI Motif in the Formation of Specific Multimeric MADS Domain Protein Complexes

The products of B class floral homeotic genes specify petal and stamen identity, and loss of B function results in homeotic conversions of petals into sepals and stamens into carpels. Here, we describe the molecular characterization of seirena-1 (sei-1), a mutant from the basal eudicot California poppy (Eschscholzia californica) that shows homeotic changes characteristic of floral homeotic B class mutants. SEI has been previously described as EScaGLO, one of four B class–related MADS box genes in California poppy. The C terminus of SEI, including the highly conserved PI motif, is truncated in sei-1 proteins. Nevertheless, like the wild-type SEI protein, the sei-1 mutant protein is able to bind CArG-boxes and can form homodimers, heterodimers, and several higher order complexes with other MADS domain proteins. However, unlike the wild type, the mutant protein is not able to mediate higher order complexes consisting of specific B, C, and putative E class related proteins likely involved in specifying stamen identity. Within the PI motif, five highly conserved N-terminal amino acids are specifically required for this interaction. Several families lack this short conserved sequence, including the Brassicaceae, and we propose an evolutionary scenario to explain these functional differences.     

Transference of function shapes organ identity in the dove tree inflorescence

? An important evolutionary mechanism shaping the biodiversity of flowering plants is the transfer of function from one plant organ to another. To investigate whether and how transference of function is associated with the remodeling of the floral organ identity program we studied Davidia involucrata, a species with conspicuous, petaloid bracts subtending a contracted inflorescence with reduced flowers. ? A detailed ontogeny enabled the interpretation of expression patterns of B-, C- and E-class homeotic MADS-box genes using qRT-PCR and in situ hybridization techniques. We investigated protein-protein interactions using yeast two-hybrid assays. ? Although loss of organs does not appear to have affected organ identity in the retained organs of the reduced flowers of D. involucrata, the bracts express the B-class TM6 (Tomato MADS box gene 6) and GLOBOSA homologs, but not DEFICIENS, and the C-class AGAMOUS homolog, representing a subset of genes also involved in stamen identity. ? Our results may illustrate how petal identity can be partially transferred outside the flower by expressing a subset of stamen identity genes. This adds to the molecular mechanisms explaining the diversity of plant reproductive morphology.     

Sex determination in the monoecious species cucumber is confined to specific floral whorls

In unisexual flowers, sex is determined by the selective repression of growth or the abortion of either male or female reproductive organs. The mechanism by which this process is controlled in plants is still poorly understood. Because it is known that the identity of reproductive organs in plants is controlled by homeotic genes belonging to the MADS box gene family, we analyzed floral homeotic mutants from cucumber, a species that bears both male and female flowers on the same individual. To study the characteristics of sex determination in more detail, we produced mutants similar to class A and C homeotic mutants from well-characterized hermaphrodite species such as Arabidopsis by ectopically expressing and suppressing the cucumber gene CUCUMBER MADS1 (CUM1). The cucumber mutant green petals (gp) corresponds to the previously characterized B mutants from several species and appeared to be caused by a deletion of 15 amino acid residues in the coding region of the class B MADS box gene CUM26. These homeotic mutants reveal two important concepts that govern sex determination in cucumber. First, the arrest of either male or female organ development is dependent on their positions in the flower and is not associated with their sexual identity. Second, the data presented here strongly suggest that the class C homeotic function is required for the position-dependent arrest of reproductive organs.     

Male and female flowers of the dioecious plant sorrel show different patterns of MADS box gene expression.

Male and female flowers of the dioecious plant sorrel (Rumex acetosa) each produce three whorls of developed floral organs: two similar whorls of three perianth segments and either six stamens (in the male) or a gynoecium consisting of a fertile carpel and two sterile carpels (in the female). In the developing male flower, there is no significant proliferation of cells in the center of the flower, in the position normally occupied by the carpels of a hermaphrodite plant. In the female flower, small stamen primordia are formed. To determine whether the organ differences are associated with differences in the expression of organ identity genes, cDNA clones representing the putative homologs of B and C function MADS box genes were isolated and used in an in situ hybridization analysis. The expression of RAD1 and RAD2 (two different DEFICIENS homologs) in males and females was confined to the stamen whorl; the lack of expression in the second, inner perianth whorl correlated with the sepaloid nature of the inner whorl of perianth segments. Expression of RAP1 (a PLENA homolog) occurred in the carpel and stamen whorls in very young flower primordia from both males and females. However, as soon as the inappropriate set of organs ceased to develop, RAP1 expression became undetectable in those organs. The absence of expression of RAP1 may be the cause of the arrest in organ development or may be a consequence.     

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.     

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.     

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.