Regulatory elements of the floral homeotic gene AGAMOUS identified by phylogenetic footprinting and shadowing

In Arabidopsis thaliana, cis-regulatory sequences of the floral homeotic gene AGAMOUS (AG) are located in the second intron. This 3-kb intron contains binding sites for two direct activators of AG, LEAFY (LFY) and WUSCHEL (WUS), along with other putative regulatory elements. We have used phylogenetic footprinting and the related technique of phylogenetic shadowing to identify putative cis-regulatory elements in this intron. Among 29 Brassicaceae species, several other motifs, but not the LFY and WUS binding sites identified previously, are largely invariant. Using reporter gene analyses, we tested six of these motifs and found that they are all functionally important for the activity of AG regulatory sequences in A. thaliana. Although there is little obvious sequence similarity outside the Brassicaceae, the intron from cucumber AG has at least partial activity in A. thaliana. Our studies underscore the value of the comparative approach as a tool that complements gene-by-gene promoter dissection but also demonstrate that sequence-based studies alone are insufficient for a complete identification of cis-regulatory sites.     

Regulatory elements controlling expression of the Drosophila homeotic gene fork head.

The region-specific homeotic gene fork head (fkh) is expressed and required in a variety of tissues of the developing Drosophila embryo. In order to identify the cis regulatory elements directing the complex spatio-temporal expression pattern of fkh, we have studied the subpatterns directed by defined fragments of fkh genomic DNA. These experiments enabled us to distinguish separate regulatory elements specific for the different expression domains of fkh. In addition, our analysis revealed several unexpected features such as the redundancy of regulatory elements and the overlap of regulatory elements with the transcribed regions of other genes. Moreover, the separation of normally contiguous elements effecting expression in the posterior terminal fkh domain appears to lead to novel expression domains which do not correspond to known developmental units in the embryo.     

Conservation of floral homeotic gene function between Arabidopsis and antirrhinum.

Several homeotic genes controlling floral development have been isolated in both Antirrhinum and Arabidopsis. Based on the similarities in sequence and in the phenotypes elicited by mutations in some of these genes, it has been proposed that the regulatory hierarchy controlling floral development is comparable in these two species. We have performed a direct experimental test of this hypothesis by introducing a chimeric Antirrhinum Deficiens (DefA)/Arabidopsis APETALA3 (AP3) gene, under the control of the Arabidopsis AP3 promoter, into Arabidopsis. We demonstrated that this transgene is sufficient to partially complement severe mutations at the AP3 locus. In combination with a weak ap3 mutation, this transgene is capable of completely rescuing the mutant phenotype to a fully functional wild-type flower. These observations indicate that despite differences in DNA sequence and expression, DefA coding sequences can compensate for the loss of AP3 gene function. We discuss the implications of these results for the evolution of homeotic gene function in flowering plants.     

Conservation of B-class floral homeotic gene function between maize and Arabidopsis

The ABC model of flower development, established through studies in eudicot model species, proposes that petal and stamen identity are under the control of B-class genes. Analysis of B- and C-class genes in the grass species rice and maize suggests that the C- and B-class functions are conserved between monocots and eudicots, with B-class genes controlling stamen and lodicule development. We have undertaken a further analysis of the maize B-class genes Silky1, the putative AP3 ortholog, and Zmm16, a putative PI ortholog, in order to compare their function with the Arabidopsis B-class genes. Our results show that maize B-class proteins interact in vitro to bind DNA as an obligate heterodimer, as do Arabidopsis B-class proteins. The maize proteins also interact with the appropriate Arabidopsis B-class partner proteins to bind DNA. Furthermore, we show that maize B-class genes are capable of rescuing the corresponding Arabidopsis B-class mutant phenotypes. This demonstrates B-class activity of the maize gene Zmm16, and provides compelling evidence that B-class gene function is conserved between monocots and eudicots.     

A novel role for the floral homeotic gene APETALA2 during Arabidopsis fruit development

The majority of the Arabidopsis fruit comprises an ovary with three primary tissue types: the valves, the replum and the valve margins. The valves, which are derived from the ovary walls, are separated along their entire length by the replum. The valve margin, which consists of a separation layer and a lignified layer, forms as a narrow stripe of cells at the valve-replum boundaries. The valve margin identity genes are expressed at the valve-replum boundary and are negatively regulated by FUL and RPL in the valves and replum, respectively. In ful rpl double mutants, the valve margin identity genes become ectopically expressed, and, as a result, the entire outer surface of the ovary takes on valve margin identity. We carried out a genetic screen in this sensitized genetic background and identified a suppressor mutation that restored replum development. Surprisingly, we found that the corresponding suppressor gene was AP2, a gene that is well known for its role in floral organ identity, but whose role in Arabidopsis fruit development had not been previously described. We found that AP2 acts to prevent replum overgrowth by negatively regulating BP and RPL, two genes that normally act to promote replum formation. We also determined that AP2 acts to prevent overgrowth of the valve margin by repressing valve margin identity gene expression. We have incorporated AP2 into the current genetic network controlling fruit development in Arabidopsis.     

Floral development and expression of floral homeotic genes are influenced by cytokinins

Tobacco plants that are somatic mosaics for the expression of a cytokinin-synthesizing gene have viviparous leaves. Epiphyllous buds can be either vegetative or floral. Floral adventitious buds can be either normal or abnormal. Abnormalities of floral development correlate with: (i) a local activation of the cytokinin-synthesizing gene, (ii) a drastic increase in floral cytokinin content, and (iii) a decrease in the steady-state levels of mRNA homologues of the homeotic genes DEFA , GLO and PLENA of Antirrhinum majus . Thus, these data show in planta that cytokinins, a class of phytohormones, are able to alter the development of floral organs and to decrease the expression of three homeotic floral genes.     

Conversion of perianth into reproductive organs by ectopic expression of the tobacco floral homeotic gene NAG1.

Mutations in the AGAMOUS (AG) gene of Arabidopsis thaliana result in the conversion of reproductive organs, stamens and carpels, into perianth organs, sepals and petals. We have isolated and characterized the putative AG gene from Nicotiana tabacum, NAG1, whose deduced protein product shares 73% identical amino acid residues with the Arabidopsis AG gene product. RNA tissue in situ hybridizations show that NAG1 RNA accumulates early in tobacco flower development in the region of the floral meristem that will later give rise to stamens and carpels. Ectopic expression of NAG1 in transgenic tobacco plants results in a conversion of sepals and petals into carpels and stamens, respectively, indicating that NAG1 is sufficient to convert perianth into reproductive floral organs.     

NTGLO: a tobacco homologue of the GLOBOSA floral homeotic gene of Antirrhinum majus: cDNA sequence and expression pattern

We report the cloning and DNA sequence of a cDNA from Nicotiana tabacum, NTGLO, as well as the pattern of expression of the NTGLO gene in wild-type tobacco plants. The NTGLO cDNA encodes a protein of 209 amino acids, which shows 73% identity with the GLO protein encoded by the GLO gene of Antirrhinum majus, a homeotic gene involved in the genetic control of flower development. Northern blot analysis shows that the NTGLO gene is expressed mainly in floral organs and, within the flower, expression is restricted to petals and stamens. The NTGLO gene most probably represents a true homologue of the GLO gene because: i) the MADS boxes, of the two genes are highly homologous (56 out of 58 amino acids are identical): ii) at the carboxyterminal a block of 19 amino acids is perfectly conserved between the NTGLO and GLO proteins and iii) their expression patterns in floral organs are identical.     

Transcript profiling of chickpea pod wall revealed the expression of floral homeotic gene AGAMOUS-like X2 (CaAGLX2)

Molecular Biology Reports - The differentially expressed genes in the chickpea pod wall have been identified for the first time using a forward suppression subtractive hybridization (SSH) library....     

The Arabidopsis floral homeotic gene APETALA3 differentially regulates intercellular signaling required for petal and stamen development

Cell-cell signaling is crucial for the coordination of cell division and differentiation during plant organogenesis. We have developed a novel mosaic analysis method for Arabidopsis, based on the maize Ac/Ds transposable element system, to assess the requirements of individual genes in intercellular signaling. Using this strategy, we have shown that the floral homeotic APETALA3 (AP3) gene has distinct roles in regulating intercellular signaling in different tissues. In petals, AP3 acts primarily in a cell-autonomous fashion to regulate cell type differentiation, but its function is also required in a non-cell-autonomous fashion to regulate organ shape. In contrast, AP3-regulated intercellular interactions are required for conferring both cell type identity and organ shape and size in the stamens. Using antibodies raised against AP3, we have shown that the AP3 protein does not traffic between cells. These observations imply that AP3 acts by differentially regulating the production of intercellular signals in a whorl-specific manner.