Uncovering genetic and molecular interactions among floral meristem identity genes in Arabidopsis thaliana

The inflorescence meristem produces floral primordia that remain undifferentiated during the first stages of flower development. Genes controlling floral meristem identity include LEAFY (LFY), APETALA1 (AP1), CAULIFLOWER (CAL), LATE MERISTEM IDENTITY 1 (LMI1), SHORT VEGETATIVE PHASE (SVP) and AGAMOUS-LIKE24 (AGL24). The lfy mutant shows partial reversions of flowers into inflorescence shoot-like structures and this phenotype is enhanced in the lfy ap1 double mutant. Here we show that combining the lfy mutant with agl24 and svp single mutants or with the agl24 svp double mutant enhances the lfy phenotype and that the lfy agl24 svp triple mutant phenocopies the lfy ap1 double mutant. Analysis of the molecular interactions between LFY, AGL24 and SVP showed that LFY is a repressor of AGL24 and SVP, whereas LMI1 is a positive regulator of these genes. Moreover, AGL24 and SVP positively regulate AP1 and LFY by direct binding to their regulatory regions. Since all these genes are important for establishing floral meristem identity, regulatory loops are probably important to maintain the correct relative expression levels of these genes.     

Identification of class B and class C floral organ identity genes from rice plants

The functions of two rice MADS-box genes were studied by the loss-of-function approach. The first gene, OsMADS4, shows a significant homology to members in the PISTILLATA (PI) family, which is required to specify petal and stamen identity. The second gene, OsMADS3, is highly homologous to the members in the AGAMOUS (AG) family that is essential for the normal development of the internal two whorls, the stamen and carpel, of the flower. These two rice MADS box cDNA clones were connected to the maize ubiquitin promoter in an antisense orientation and the fusion molecules were introduced to rice plants by the Agrobacterium-mediated transformation method. Transgenic plants expressing antisense OsMADS4 displayed alterations of the second and third whorls. The second-whorl lodicules, which are equivalent to the petals of dicot plants in grasses, were altered into palea/lemma-like organs, and the third whorl stamens were changed to carpel-like organs. Loss-of-function analysis of OsMADS3 showed alterations in the third and fourth whorls. In the third whorl, the filaments of the transgenic plants were changed into thick and fleshy bodies, similar to lodicules. Rather than making a carpel, the fourth whorl produced several abnormal flowers. These phenotypes are similar to those of the agamous and plena mutants in Arabidopsis and Antirrhinum, respectively. These results suggest that OsMADS4 belongs to the class B gene family and OsMADS3 belongs to the class C gene family of floral organ identity determination.     

On the origin of class B floral homeotic genes: functional substitution and dominant inhibition in Arabidopsis by expression of an orthologue from the gymnosperm Gnetum

Class B floral homeotic genes are involved in specifying stamen and petal identity in angiosperms (flowering plants). Here we report that gymnosperms, the closest relatives of the angiosperms, contain at least two different clades representing putative orthologues of class B genes, termed GGM2-like and DAL12-like genes. To obtain information about the functional conservation of the class B genes in seed plants, the representative of one of these clades from Gnetum, termed GGM2, was expressed under the control of the CaMV 35S promoter in Arabidopsis wild-type plants and in different class B mutants. In wild-type plants and in a conditional mutant grown at a permissive temperature, gain-of-function phenotypes were obtained in whorls 1 and 4, where class B genes are usually not expressed. In contrast, loss-of-function phenotypes were observed in whorls 2 and 3, where class B genes are expressed. In different class B gene null mutants of Arabidopsis, and in the conditional B mutant grown at the non-permissive temperature, a partial complementation of the mutant phenotype was obtained. In situ hybridization studies and class B gene promoter test fusion experiments demonstrated that the gain-of-function phenotypes are not due to an upregulation of the endogenous B genes from Arabidopsis, and hence probably involve interactions between GGM2 protein homodimers and class B protein target genes other than the Arabidopsis class B genes itself. To our knowledge, this is the first time that partial complementation of a homeotic mutant by an orthologous gene from a distantly related species has been reported. These data suggest that GGM2 has a function in the gymnosperm Gnetum which is related to that of class B floral organ identity genes of angiosperms. That function may be in the specification of male reproductive organ identity, and in distinguishing male from female reproductive organs.     

Structural basis for LEAFY floral switch function and similarity with helix-turn-helix proteins

The LEAFY (LFY) protein is a key regulator of flower development in angiosperms. Its gradually increased expression governs the sharp floral transition, and LFY subsequently controls the patterning of flower meristems by inducing the expression of floral homeotic genes. Despite a wealth of genetic data, how LFY functions at the molecular level is poorly understood. Here, we report crystal structures for the DNA-binding domain of Arabidopsis thaliana LFY bound to two target promoter elements. LFY adopts a novel seven-helix fold that binds DNA as a cooperative dimer, forming base-specific contacts in both the major and minor grooves. Cooperativity is mediated by two basic residues and plausibly accounts for LFY's effectiveness in triggering sharp developmental transitions. Our structure reveals an unexpected similarity between LFY and helix-turn-helix proteins, including homeodomain proteins known to regulate morphogenesis in higher eukaryotes. The appearance of flowering plants has been linked to the molecular evolution of LFY. Our study provides a unique framework to elucidate the molecular mechanisms underlying floral development and the evolutionary history of flowering plants.     

The cycloidea gene can respond to a common dorsoventral prepattern in Antirrhinum

Dorsoventral asymmetry in flowers of Antirrhinum depends on expression of the cycloidea gene in dorsal regions of floral meristems. To determine how cycloidea might be regulated we analysed its expression in several contexts. We show that cycloidea is activated shortly after floral induction, and that in addition to flowers, cycloidea can be asymmetrically expressed in shoots, even though these shoots show no marked dorsoventral asymmetry. Shoots expressing cycloidea include secondary branches lying just below the inflorescence, and shoots of floricaula mutants. Asymmetric cycloidea expression may also be observed within organ primordia, such as the sepals of terminal flowers produced by centroradialis mutants. Later expression of cycloidea within flowers can be modified by mutations in organ identity genes. Taken together, the results suggest that cycloidea can respond to a common dorsoventral pre-pattern in the apex and that the specific effects of cycloidea on the flower depend on interactions with floral-specific genes.     

Flower-specific expression of the Agrobacterium tumefaciens isopentenyltransferase gene results in radial expansion of floral organs in Petunia hybrida

Biotechnology has the potential to modify commercially important traits of crops, such as fruit size and stress tolerance. To date, the floricultural industry has not profited significantly from these possibilities to manipulate, for example, flower size. Cytokinins are known to be involved in many aspects of plant development, including cell division. Increasing the amount of cytokinins has the potential to increase the size of an organ, such as the flower or the fruit. The Agrobacterium tumefaciens cytokinin biosynthesis gene isopentenyltransferase ( ipt ) has been shown to increase cytokinin levels when introduced into plants. Moreover, it has a dramatic effect on the vegetative development of plants. The expression of the ipt gene under the control of the flower-specific Arabidopsis APETALA3 promoter in petunia ( Petunia hybrida ) increases the flower size dramatically, but with no effect on vegetative development. The resulting transgenic plants produced flowers with larger corolla diameter and greater total floral fresh weight. This strategy has the potential for use in the production of ornamental crops with large flowers and crop species with larger fruit.     

Genetic complementation of a floral homeotic mutation,apetala3, with an Arabidopsis thaliana gene homologous to DEFICIENS of Antirrhinum majus

Among the homeotic mutants with altered floral organs, two mutants of Arabidopsis thaliana, apetala3 and pistillata, and two mutants of Antirrhinum majus, deficiens and globosa, have a homeotic conversion of the floral organs in whorl 2 and 3, namely petals to sepals and stamens to carpels. We have isolated a homologue of the DEFICIENS gene from A. thaliana wild type and shown complete complementation of apetala3 mutation by introducing the isolated gene using Agrobacterium-mediated transformation. These results show that the APETALA3 is a homologue of DEFICIENS structurally and functionally. The 5-upstream region of APETALA3 contains three SRE-like sequence, where MADS box-containing proteins are assumed to bind and regulate expression in tissue-and stage-specific manner.     

The molecular population genetics of regulatory genes

Regulatory loci, which may encode both trans acting proteins as well as cis acting promoter regions, are crucial components of an organism's genetic architecture. Although evolution of these regulatory loci is believed to underlie the evolution of numerous adaptive traits, there is little information on natural variation of these genes. Recent molecular population genetic studies, however, have provided insights into the extent of natural variation at regulatory genes, the evolutionary forces that shape them and the phenotypic effects of molecular regulatory variants. These recent analyses suggest that it may be possible to study the molecular evolutionary ecology of regulatory diversification by examining both the extent and patterning of regulatory gene diversity, the phenotypic effects of molecular variation at these loci and their ecological consequences.     

Unexpected behavior of a gene trap vector comprising a fusion between the Sh ble and the lacZ genes

A new gene trap vector has been designed, comprised of a fusion between the Sh ble gene, which confers resistance to the antibiotic phleomycin, and the lacZ gene (phleal fusion gene). A synthetic splice acceptor, made of the yeast branchpoint followed by a pyrimidin-rich sequence of 27 nucleotides, is included at the 5′ extremity. The linearized gene trap vector was introduced into mouse embryonic stem cells (ES cells), and 40 phleomycin resistant (phleAL) cell lines possessing a single copy of the insert were selected. They were stable in expressing the lacZ gene. Reporter gene expression was studied at days 8.5 and 10.5 of embryonic development in chimeric embryos obtained after injection of phleoL ES clones into 8-cell stage embryos. Out of 20 phleal lines examined, 14 exhibited β-galactosidase expression at day 10.5. Use of the phleal fusion gene trap vector to select genes expressed in ES cells, therefore, is compatible with the isolation of genes expressed at midgestation. However, and most intriguingly, 10 out of these 14 cell lines (71%) displayed reporter gene expression mostly in heart and liver. Two of them exhibited, in addition, expression in central nervous system (CNS) or in CNS and limb buds, respectively. Germline chimeras were subsequently obtained and 15 mouse lines have been established. Intercrosses of animals heterozygous for the insertion revealed a mutant phenotype in several lines. © 1996 Wiley-Liss, Inc.