GEG participates in the regulation of cell and organ shape during corolla and carpel development in gerbera hybrida

The molecular mechanisms that control organ shape during flower development are largely unknown. By using differential hybridization techniques, a cDNA designated GEG (for Gerbera hybrida homolog of the gibberellin [GA]-stimulated transcript 1 [GAST1] from tomato) was isolated from a library representing late stages of corolla development in Gerbera. GEG expression was detected in corollas and carpels, with expression spatiotemporally coinciding with flower opening. In corollas and styles, GEG expression is temporally correlated with the cessation of longitudinal cell expansion. In plants constitutively expressing GEG, reduced corolla lengths and carpels with shortened and radially expanded stylar parts were found, with concomitant reduction of longitudinal cell expansion in these organs. In addition, in styles, an increase in radial cell expansion was detected. Taken together, these observations indicate a regulatory role for the GEG gene product in determining the shape of the corolla and carpel. The deduced amino acid sequence of the GEG gene product shares high similarity with previously characterized putative cell wall proteins encoded by GA-inducible genes, namely, GAST1, GIP (for GA-induced gene of petunia), and the GASA (for GA-stimulated in Arabidopsis) gene family. Our studies suggest that GEG, the expression of which can also be induced by application of GA3, plays a role in phytohormone-mediated cell expansion.

Organ identity genes and modified patterns of flower development in Gerbera hybrida (Asteraceae)

We have used Gerbera hybrida (the cultivated ornamental, gerera) to investigate the molecular basis of flower development in Asteraceae, a family of flowering plants that have heteromorphic flowers and specialized floral organs. Flowers of the same genotype may differ in a number of parameters, including sex expression, symmetry, sympetaly and pigmentation. In order to study the role of organ identity determination in these phenomena we isolated and functionally analysed six MADS box genes from gerbera; these were shown by phylogenetic analysis to be orthologous to well characterized regulatory genes described from Arabidopsis and Antirrhinum. Expression analysis suggests that the two gerbera agamous orthologues, the globosa orthologue and one of the deficiens orthologues may have functional equivalency to their counterparts, participating in the C and B functions, respectively. However, the function of a second deficiens orthologue appears unrelated to the B function, and that of a squamosa orthologue seems distinct from squamosa as well as from the A function. The induction patterns of gerbera MADS box genes conform spatiotemporally to the multi-flowered, head-like inflorescence typical of Asteraceae. Furthermore, gerbera plants transgenic for the newly isolated MADS box genes shed light onto the mechanistic basis for some floral characteristics that are typical for Asteraceae. We can conclude, therefore, that the pappus bristles are sepals highly modified for seed dispersal, and that organ abortion in the female marginal flowers is dependent upon organ identity and not organ position when position is homeotically altered.

A bHLH transcription factor mediates organ, region and flower type specific signals on dihydroflavonol-4-reductase (dfr) gene expression in the inflorescence of Gerbera hybrida (Asteraceae).

The angiosperm family Asteraceae is characterized by composite inflorescences, which are highly organized structures consisting of different types of flowers. In order to approach the control of floral organ differentiation in Asteraceae at molecular level, we are studying regulation of flavonoid biosynthesis in Gerbera hybrida. Dihydroflavonol-4-reductase (dfr) expression is regulated according to anthocyanin pigmentation patterns in all tested gerbera varieties at several anatomical levels. We have isolated a promoter for one of the dfr genes, Pgdfr2. Gerbera plants transgenic for a Pgdfr2-uidA construct reveal that the activity of the Pgdfr2 promoter from one variety follows the pigmentation in other varieties which have different color patterns. It is thus evident that the observed complex regulation of dfr expression occurs in trans. In order to identify the trans-acting regulators, we isolated a cDNA (gmyc1) homologous to the previously characterized genes encoding bHLH-type regulators of the anthocyanin pathway in plants. The expression of gmyc1 in different varieties suggests that it has a major role in regulating dfr activity in corolla and carpel, but not in pappus and stamen. Specifically in gerbera, the identical patterns of gmyc1 and dfr expression in corolla tissue suggest that GMYC1 also regulates dfr expression in a region and flower type specific manner. Our studies show that in gerbera GMYC1-dfr interaction is part of several developmental processes characteristic for Asteraceae (such as specification of flower types across the composite inflorescence), whereas in other processes (such as differentiation of sepal as pappus) other regulators control dfr expression to determine the spatial specificity.

Analysis of androgen receptor-DNA interactions with receptor proteins produced in insect cells.

Wild-type rat androgen receptor and four of its deletion mutants were produced in insect cells using the baculovirus expression system. Inclusion of androgen, but not estrogen, progesterone, or glucocorticoid, in culture medium increased the yield of soluble androgen receptors, although the majority of receptors still remained in the insoluble form (Xie, Y.-B., Sui, Y.-P., Shan, L.-X., Palvimo, J. J., Phillips, D. M., and Jänne, O. A. (1992) J. Biol. Chem. 267, 4939-4948). The wild-type receptor interacted with an androgen response element (ARE) with a 2-6-fold higher affinity (KD = 0.5 nM) than mutants with deletions outside the DNA-binding domain (delta 40-147, delta 38-296, delta 46-408, and delta 788-902 mutants), suggesting that sequences flanking the DNA-binding region influence the stability of receptor-DNA complexes. Changes in spacing (n = 3) between the two ARE half-sites by a single nucleotide (n - 1, n + 1) or by 10 bases (n + 10) abolished the full-length receptor's ability to form stable complexes with DNA. Binding to AREs with altered spacing could not be restored by antisera against the N-terminal domain of the receptor that stabilize androgen receptor-DNA interactions with many naturally occurring strong and weak AREs. Methylation interference and 1,10-phenanthroline copper footprinting analyses revealed that the receptor binds to DNA as a dimer. Dimer formation was demonstrated directly by mixing full-length and delta 46-408 mutant receptors, which resulted in the formation of heterodimeric receptor-DNA complexes. The half-time of dissociation of the wild-type receptor from a consensus ARE sequence was about 3 min at 22 degrees C. Collectively, androgen receptor binds to DNA with properties similar to, but not identical with, those of glucocorticoid receptor, indicating that regions outside the DNA-binding domain are important to ensure androgen specificity of transcriptional regulation.

Dominant negative regulation of trans-activation by the rat androgen receptor: roles of the N-terminal domain and heterodimer formation.

A series of deletion mutants was constructed for the rat androgen receptor (AR) to delineate sequences involved in transcriptional activation. Using transient expression conditions in CV-1 cells and in vitro DNA-binding studies, the amino-terminal domain of the receptor was shown to contain a region (residues 147-296) that is mandatory for trans-activation. Receptors with deletions (residues 147-408) in the N-terminal domain, but with intact DNA- and ligand-binding domains, interacted in vitro with androgen-responsive elements albeit with affinities lower than that of the wild type receptor. Coexpression of N-terminal deletion mutants (delta 46-408 and delta 38-296) with the wild type AR blunted trans-activation by the latter protein in a dominant fashion. By contrast, a hormone-binding-deficient receptor (delta 788-902) that had poor intrinsic activity potentiated the trans-activation by the native receptor. Mechanisms by which deletion mutants in the N-terminal region abolish the function of the wild type protein appear to involve heterodimer formation during interaction with DNA and direct competition for available binding sites on DNA, rather than squelching of accessory proteins. In contrast to impaired trans-activation, binding of the ligand to N-terminal deletion mutants brought about conformational changes that were comparable in wild type and mutant forms, as judged by electrophoretic mobility shift assays. Taken together, these data have specified a region in the N-terminal domain of the AR that plays a decisive role in transcriptional regulation.

Transgenic potato plants expressing mammalian 2'-5' oligoadenylate synthetase are protected from potato virus X infection under field conditions.

We cloned and sequenced a rat cDNA encoding the 2'-5' oligoadenylate synthetase, a component of the mammalian interferon-induced antiviral response, and used Agrobacterium-mediated transformation to generate transgenic potato clones expressing this mammalian enzyme. In transgenic plants infected with potato virus X and followed under field conditions, virus concentrations in leaves and in tubers were significantly lower than in nontransgenic controls. Additionally, virus concentration in the leaves of five transgenic clones and in tubers of one clone was also lower than in transgenic potatoes expressing potato virus X coat protein.

Production of recombinant androgen receptor in a heterologous expression system.

To facilitate detailed studies of androgen receptor, we have produced a full-length receptor protein and some of its deletion mutants in Spodoptera frugiperda (Sf9) insect cells, using the baculovirus expression system. Recombinant baculovirus DNA-infected Sf9 cells expressed these proteins in very high quantities, which represented as much as 30-40% of total insect cell protein at 72 h after infection. Only < 1% of the recombinant protein was soluble in low-salt buffers; the majority formed electron-dense cytoplasmic aggregates 30-40 nm in diameter. These aggregates could be solubilized in 6 mol/L guanidine HCl, and biologically active receptor was generated by diluting the guanidine HCl preparation 20- to 50-fold. The full-length receptor, expressed either in a soluble or aggregated form, had characteristics typical of a native receptor: it bound steroids with high affinity and specificity, interacted with DNA in a sequence-specific fashion, and was recognized by domain-specific receptor antibodies. Androgen-receptor protein purified to homogeneity in guanidine HCl required the presence of Zn2+ ions during the refolding to reconstitute its DNA-binding form; ZnCl2 was not, however, needed to restore the receptor's steroid-binding activity.

Androgen receptor and mechanism of androgen action.

Androgen receptor is the intracellular protein that mediates biological actions of physiological androgens (testosterone and 5 alpha-dihydrotestosterone). Androgen receptor belongs to a large family of ligand-dependent proteins whose function is to modulate expression of genes and gene networks in a cell- and tissue-specific manner. The present overview describes the structurally important domains of the receptor protein, and discusses several aspects in the structure-function relationship, using naturally occurring receptor mutants in androgen insensitivity patients or experimental animals as examples. In addition, characteristics of androgen receptor expressed in a heterologous system are described, and their potential usefulness in specific molecular studies discussed.