Molecular and expression analysis of a LIM protein gene family from flowering plants.

LIM-domain proteins participate in important cellular processes in eukaryotes, including gene transcription and actin cytoskeleton organization. They are predominantly found in animals, but have also been identified in yeast and plants. Following the characterization ofa LIM-domain protein in sunflower pollen, we carried out an extensive search for these proteins in flowering plants. We have isolated and studied cDNAs and/or genomic sequences for two novel LIM-domain proteins from sunflower, three from tobacco, and one from Arabidopsis. The plant proteins are structurally related to the cytoskeleton-associated CRP class of LIM proteins in animals, but show several distinctive features, including a second, atypical, LIM domain. We have performed comparative expression studies of these genes, as well as of one other gene from tobacco and two additional Arabidopsis genes whose sequences are available from databases. These studies, carried out by RT-PCR in the presence of gene-specific primers, showed that, in sunflower and tobacco, pollen grains and sporophytic tissues express different sets of LIM proteins. With the exception of one Arabidopsis gene--which has two introns--all the genes analyzed contain four introns at conserved positions, indicating that the ancestral gene from which the various copies evolved in higher plants allready had this split structure.

A LIM-domain protein from sunflower is localized to the cytoplasm and/or nucleus in a wide variety of tissues and is associated with the phragmoplast in dividing cells.

LIM proteins are important eucaryotic developmental regulators characterized by the presence of one or several double zinc finger motifs, the LIM domains, which are protein-interacting domains. Using the cDNA of the previously described pollen LIM protein PLIM1 from sunflower as a hybridization probe we have isolated the coding sequence for a related protein from cDNA libraries from various sunflower organs. This protein, WLIM1, is 188 amino acids long and, like the pollen protein PLIM1, contains two LIM domains, separated by a 48 residue spacer region. The two sunflower proteins are structurally related to the animal LIM proteins CRP and MLP. A WLIM1 gene transcript was detected by RT-PCR in all vegetative and reproductive plant organs tested. Polyclonal antibodies raised against the bacterially expressed and affinity-purified protein recognize a polypeptide of ca. 50 kDa in these organs. Immunocytochemical studies detect the protein in many cell types in each of these organs where it is localized either to the cytoplasm, the nucleus, or both. The protein is often associated with plastids and smaller cellular structures or organelles. In late anaphase and early telophase of dividing cells from ovaries, stems and roots it accumulates in the phragmoplast, and may therefore also play a role in cytokinesis.

Antifungal activity of a virally encoded gene in transgenic wheat.

The cDNA encoding the antifungal protein KP4 from Ustilago maydis-infecting virus was inserted behind the ubiquitin promoter of maize and genetically transferred to wheat varieties particularly susceptible to stinking smut (Tilletia tritici) disease. The transgene was integrated and inherited over several generations. Of seven transgenic lines, three showed antifungal activity against U. maydis. The antifungal activity correlated with the presence of the KP4 transgene. KP4-transgenic, soil-grown wheat plants exhibit increased endogenous resistance against stinking smut.

Interferometric sensor for detection of surface-bound bioreactions.

An integrated optical interferometer for direct detection of affinity reactions is presented. A modern version of a Young's interferometer is built with a waveguide structure as beam splitter and as sensing element. Resistive waveguides were produced by plasma-enhanced chemical vapor deposition of silicon oxinitride. At the output of this device a fringe pattern is detected by a CCD line camera. The adsorption of molecules on top of the waveguides is observed with a detection limit of 750 fg/mm(2). The resolvable variation of effective refractive index is 9 x 10(-8).

Efficient interspecific hybridization in the genusHelianthus via "embryo rescue" and characterization of the hybrids.

With the aid of the "embryo rescue" technique, interspecific hybrids in the genusHelianthus could be raised with a recovery rate of 41%. Altogether, 33 different hybrid combinations were realized using the cultivated form, both as a female and male parent. The hybrids obtained have been identified by different methods, i.e., by comparison of leaf morphology, pollen stainability, chromosome number and by RFLP analysis. The former three methods are useful to obtain global information, while the RFLP analysis allows a rapid and safe characterization in early developmental stages of the hybrids.