Ternary complex formation between the MADS-box proteins SQUAMOSA, DEFICIENS and GLOBOSA is involved in the control of floral architecture in Antirrhinum majus.

In Antirrhinum, floral meristems are established by meristem identity genes. Floral meristems give rise to floral organs in whorls, with their identity established by combinatorial activities of organ identity genes. Double mutants of the floral meristem identity gene SQUAMOSA and organ identity genes DEFICIENS or GLOBOSA produce flowers in which whorled patterning is partially lost. In yeast, SQUA, DEF and GLO proteins form ternary complexes via their C-termini, which in gel-shift assays show increased DNA binding to CArG motifs compared with DEF/GLO heterodimers or SQUA/SQUA homodimers. Formation of ternary complexes by plant MADS-box factors increases the complexity of their regulatory functions and might be the molecular basis for establishment of whorled phyllotaxis and combinatorial interactions of floral organ identity genes.     

To B or Not to B a flower: the role of DEFICIENS and GLOBOSA orthologs in the evolution of the angiosperms

DEFICIENS (DEF) and GLOBOSA (GLO) function in petal and stamen organ identity in Antirrhinum and are orthologs of APETALA3 and PISTILLATA in Arabidopsis. These genes are known as B-function genes for their role in the ABC genetic model of floral organ identity. Phylogenetic analyses show that DEF and GLO are closely related paralogs, having originated from a gene duplication event after the separation of the lineages leading to the extant gymnosperms and the extant angiosperms. Several additional gene duplications followed, providing multiple potential opportunities for functional divergence. In most angiosperms studied to date, genes in the DEF/GLO MADS-box subfamily are expressed in the petals and stamens during flower development. However, in some angiosperms, the expression of DEF and GLO orthologs are occasionally observed in the first and fourth whorls of flowers or in nonfloral organs, where their function is unknown. In this article we review what is known about function, phylogeny, and expression in the DEF/GLO subfamily to examine their evolution in the angiosperms. Our analyses demonstrate that although the primary role of the DEF/GLO subfamily appears to be in specifying the stamens and inner perianth, several examples of potential sub- and neofunctionalization are observed.     

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.     

Evolution and function of MADS‐box genes involved in orchid floral development

Orchids are known for their beauty and complexity of flower and ecological strategies. The evolution in orchid floral morphology, structure, and physiological properties has held the fascination of botanists for centuries, from Darwin through to the present. In floral studies, MADS‐box genes contributing to the now famous ABCDE model of floral organ identity control have dominated conceptual thinking. The sophisticated orchid floral organization offers an opportunity to discover new variant genes and different levels of complexity to the ABCDE model. Recently, several remarkable research reports on orchid MADS‐box genes, especially B‐class MADS‐box genes, have revealed the evolutionary track and important functions on orchid floral development. Diversification and fixation of both paleoAP3 gene sequences and expression profiles might be explained by subfunctionalization and even neofunctionalization. Knowledge about MADS‐box genes encoding ABCDE functions in orchids will give insights into the highly evolved floral morphogenetic networks of orchids.     

The K domain mediates heterodimerization of the Arabidopsis floral organ identity proteins,APETALA3 and PISTILLATA

MADS genes in plants encode key developmental regulators of vegetative and reproductive development. The majority of well-characterized plant MADS proteins contain two conserved domains, the DNA-binding MADS domain and the K domain. The K domain is predicted to form three amphipathic alpha-helices referred to as K1, K2, and K3. In this report, we define amino acids and subdomains important for heterodimerization between the two Arabidopsis floral organ identity MADS proteins APETALA3 (AP3) and PISTILLATA (PI). Analysis of mutants defective in dimerization demonstrates that K1, K2 and the region between K1 and K2 are critical for the strength of AP3/PI dimerization. The majority of the critical amino acids are hydrophobic indicating that the K domain mediates AP3/PI interaction primarily through hydrophobic interactions. Specially, K1 of AP3 and PI resembles a leucine zipper motif. Most mutants defective in AP3/PI heterodimerization in yeast exhibit partial floral organ identity function in transgenic Arabidopsis. Our results also indicate that the motif containing Asn-98 and specific charged residues in K1 (Glu-97 in PI and Arg-102 in AP3) are important for both the strength and specificity of AP3/PI heterodimer formation.     

The Arabidopsis AGL9 MADS box gene is expressed in young flower primordia

 MADS box genes are likely involved in many different steps of plant development, since their RNAs accumulate in a wide variety of tissues, including roots, stems, leaves, flowers and embryos. In flowers, MADS box genes regulate the early step of specifying floral meristem identity as well as the later step of determining the fate of floral organ primordia. Here we describe the isolation and characterization of a new MADS box gene from Arabidopsis, AGL9. Sequence analyses indicate that AGL9 represents the putative ortholog of the FBP2 and TM5 genes from petunia and tomato, respectively. In situ hybridization analyses show that AGL9 RNA begins to accumulate after the onset of expression of the floral meristem identity genes, but before the activation of the organ identity genes. These data indicate that AGL9 functions early in flower development to mediate between the interaction of these two classes of genes. Later in flower development, AGL9 RNA accumulates in petals, stamens, and carpels, suggesting a role for AGL9 in controlling the development of these organs. Received: 4 May 1997 / Accepted: 14 July 1997     

AGL24, SHORT VEGETATIVE PHASE, and APETALA1 redundantly control AGAMOUS during early stages of flower development in Arabidopsis

Loss-of-function alleles of AGAMOUS-LIKE24 (AGL24) and SHORT VEGETATIVE PHASE (SVP) revealed that these two similar MADS box genes have opposite functions in controlling the floral transition in Arabidopsis thaliana, with AGL24 functioning as a promoter and SVP as a repressor. AGL24 promotes inflorescence identity, and its expression is downregulated by APETALA1 (AP1) and LEAFY to establish floral meristem identity. Here, we combine the two mutants to generate the agl24 svp double mutant. Analysis of flowering time revealed that svp is epistatic to agl24. Furthermore, when grown at 30 degrees C, the double mutant was severely affected in flower development. All four floral whorls showed homeotic conversions due to ectopic expression of class B and C organ identity genes. The observed phenotypes remarkably resembled the leunig (lug) and seuss (seu) mutants. Protein interaction studies showed that dimers composed of AP1-AGL24 and AP1-SVP interact with the LUG-SEU corepressor complex. We provide genetic evidence for the role of AP1 in these interactions by showing that the floral phenotype in the ap1 agl24 svp triple mutant is significantly enhanced. Our data suggest that MADS box proteins are involved in the recruitment of the SEU-LUG repressor complex for the regulation of AGAMOUS.     

Diverse roles for MADS box genes in Arabidopsis development.

Members of the MADS box gene family play important roles in flower development from the early step of determining the identity of floral meristems to specifying the identity of floral organ primordia later in flower development. We describe here the isolation and characterization of six additional members of this family, increasing the number of reported Arabidopsis MADS box genes to 17. All 11 members reported prior to this study are expressed in flowers, and the majority of them are floral specific. RNA expression analyses of the six genes reported here indicate that two genes, AGL11 and AGL13 (AGL for AGAMOUS-like), are preferentially expressed in ovules, but each has a distinct expression pattern. AGL15 is preferentially expressed in embryos, with its onset at or before the octant stage early in embryo development. AGL12, AGL14, and AGL17 are all preferentially expressed in root tissues and therefore represent the only characterized MADS box genes expressed in roots. Phylogenetic analyses showed that the two genes expressed in ovules are closely related to previously isolated MADS box genes, whereas the four genes showing nonfloral expression are more distantly related. Data from this and previous studies indicate that in addition to their proven role in flower development, MADS box genes are likely to play roles in many other aspects of plant development.     

APETALA3/DEFICIENS和PISTILLATA/GLOBOSA基因与植物花发育

APETALA3(AP3)/DEFICIENS(DEF)和PISTILLATA(PI)/GLOBOSA(GLO)为植物花器官发育B类基因,控制双子叶植物花瓣和雄蕊的发育,它们属于MADS-box基因家族,编码转录因子,这些基因的突变能导致花瓣转变为萼片,雄蕊转变为心皮。近年来已经在多种植物中克隆到了AP3/DEF和PI/GLO基因,AP3/DEF和PI/GLO基因在拟南芥中只在花器官中表达,而在玉米等植物维管束、叶片等组织中也有表达。现对有关AP3/DEF和PI/GLO基因表达及其在植物系统发育学研究方面的进展进行综述。     

Alteration of tobacco floral organ identity by expression of combinations of Antirrhinum MADS-box genes

Floral organ identity is largely controlled by the spatially restricted expression of several MADS-box genes. In Antirrhinum majus these organ identity genes include DEF, GLO and PLE . Single and double mutant analyses indicated that the type of organ found in a particular whorl is dependent on which combination of these genes is expressed there. This paper reports the ectopic expression of Antirrhinum organ identity genes, alone and in combinations, in transgenic tobacco. Although the phenotypes are broadly in agreement with the genetic predictions, several unexpected features are observed which provide information concerning the action of the organ identity genes. The presumed tobacco homologue of DEF, NTDEF , has been isolated and used to investigate the influence of ectopic expression of the Antirrhinum organ identity genes on the endogenous tobacco genes. Analysis of the spatial and temporal expression patterns of NTDEF and NTGLO reveals that the boundaries are not coincident and that differences exist in the regulatory mechanisms of the two genes concerning both induction and maintenance of gene expression. Evidence is provided which indicates that organ development is sensitive to the relative levels of organ identity gene expression. Expression of the organ identity genes outside the flower or inflorescence produced no effects, suggesting that additional factors are required to mediate their activity. These results demonstrate that heterologous genes can be used to predictably influence floral organ identity but also reveal the existence of unsuspected control mechanisms.     

Cloning and characterization of four apple MADS box genes isolated from vegetative tissue

With the aim of finding genes involved in the floral transition of woody species four MADS box genes containing cDNAs from apple (Malus domestica) have been isolated. Three genes were isolated from vegetative tissue of apple, but were homologues of known genes that specify floral organ identity. MdMADS13 is an AP3-like B class MADS box gene, and was mainly expressed in petals and stamens as demonstrated by Northern blot analysis. MdMADS14 and -15 are AGAMOUS-like genes. They differed slightly in expression patterns on Northern blots, with MdMADS15 mRNA levels equally high in stamens and carpels, but MdMADS14 preferably expressed in carpels. MdMADS14 is likely to be the apple orthologue of one of the Arabidopsis thaliana SHATTERPROOF genes, and MdMADS15 closely resembled the Arabidopsis AGAMOUS gene. It has been shown with RT-PCR that the three floral apple MADS box genes are expressed in vegetative tissues of adult as well as juvenile trees, albeit at low levels. MdMADS12 is an AP1-like gene that is expressed at similar levels in leaves, vegetative shoots, and floral tissues, and that may be involved in the transition from the juvenile to the adult stage.     

Analysis of the Petunia TM6 MADS box gene reveals functional divergence within the DEF/AP3 lineage

Antirrhinum majus DEFICIENS (DEF) and Arabidopsis thaliana APETALA3 (AP3) MADS box proteins are required to specify petal and stamen identity. Sampling of DEF/AP3 homologs revealed two types of DEF/AP3 proteins, euAP3 and TOMATO MADS BOX GENE6 (TM6), within core eudicots, and we show functional divergence in Petunia hybrida euAP3 and TM6 proteins. Petunia DEF (also known as GREEN PETALS [GP]) is expressed mainly in whorls 2 and 3, and its expression pattern remains unchanged in a blind (bl) mutant background, in which the cadastral C-repression function in the perianth is impaired. Petunia TM6 functions as a B-class organ identity protein only in the determination of stamen identity. Atypically, Petunia TM6 is regulated like a C-class rather than a B-class gene, is expressed mainly in whorls 3 and 4, and is repressed by BL in the perianth, thereby preventing involvement in petal development. A promoter comparison between DEF and TM6 indicates an important change in regulatory elements during or after the duplication that resulted in euAP3- and TM6-type genes. Surprisingly, although TM6 normally is not involved in petal development, 35S-driven TM6 expression can restore petal development in a def (gp) mutant background. Finally, we isolated both euAP3 and TM6 genes from seven solanaceous species, suggesting that a dual euAP3/TM6 B-function system might be the rule in the Solanaceae.