Immunolocalization of the petunia floral binding proteins 7 and 11 during seed development in wild-type and expression mutants ofPetunia hybrida

Abstract

Summary. During Petunia hybrida seed development, the MADSbox genes encoding the floral binding proteins (FBP) 7 and 11 are expressed in the seed coat and not in the endosperm or embryo. These proteins are thought to function as transcription factors and are essential for ovule formation in Petunia spp. Immunocytochemical methods were used to analyze the distribution of FBP7 and FBPll after fertilization in wild type and ectopic and cosuppression mutants. During the first nine days of seed development the protein was found in the nuclei of seed coat cells, of both wild-type plants and plants which ectopically expressed FBPll. The signal for FBP7 and -11 proteins diminished during seed development, was first lost in the outer epidermis of the seed coat, then in the endothelium, and finally, at 9 days after pollination (DAP), the protein could not be detected anymore in the parenchyma ceils of the seed coat. Although the distribution patterns in wild-type and FBPll ectopically expressing plants are similar, the latter exhibited higher protein levels. A mild-cosuppression mutant of FBP7 and FBPll, having only a total of 5% FBP7 and -11 mRNA, showed hardly any FBP7 and -11 proteins.The lack of FBP7 and -11 caused endosperm degeneration in the mutant at a moment when the protein had already decreased to an undetectable level in the wild type and ectopic expression mutant (i.e., at 13 DAP). It is suggested that till about 9 DAP a minimal amount of FBP7 and -11 is needed for the normal functioning of the seed coat during later stages, i.e., for transfer of nutrients to endosperm and embryo. Besides the immunocytochemical data on the FBP7 and FBPll MADS-box gene products, the morphological analysis of wild type and mutants contributes details on early seed development in Petunia hybrida.

Temporal and Spatial Expression of MADS Box Genes, FBP7 and FBP11, During Initiation and Early Development of Ovules in Wild Type and Mutant Petunia hybrida

“…Recently, Wittich et al analysed the post fertilization development of Petunia seeds and demonstrated that FBP7/11 proteins are present in the cell nuclei. This nuclear position is in agreement with their supposed role as transcription factors (Wittich et al, 1999 [18] ).…”

“…In addition, parts of sepals with ovule-like structures were dissected out. Further processing was as described by Wittich et al (1999 [18] ). In short, samples were rapidly freezefixed in liquid propane, the frozen samples were freeze-substituted in acetone containing 1 % glutaradehyde in a freeze-substitution machine, further dehydrated through an acetone± ethanol series, and infiltrated and embedded in butyl methyl methacrylate (BMM).…”

Condensin and cohesin knockouts in Arabidopsis exhibit a titan seed phenotype

“…Seeds prepared for visualization of microtubules were cryo‐fixed, freeze‐substituted, and embedded in butyl methyl methacrylate (Wittich et al ., 1999). Sections 2–3 µm in thickness were washed in acetone, blocked, and labeled with monoclonal anti‐α‐tubulin (1 : 100, Sigma) and goat antimouse antibody (GaM‐IgG) conjugated to Alexa 488 (1 : 200, Molecular Probes, Leiden, The Netherlands).…”

The Last Step of Syringyl Monolignol Biosynthesis in Angiosperms Is Regulated by a Novel Gene Encoding Sinapyl Alcohol Dehydrogenase

“…After three washes with water, the sections were incubated in 4% sodium sulfite at room temperature for 5 min, mounted in 50% glycerol, and photographed using a Nikon (Tokyo, Japan) Eclipse 400 fluorescence microscope. Segments ‫1ف(‬ mm thick) from the same internodes used for histochemical analysis were used for immunolocalization on the basis of the protocol of Wittich et al (1999) with modifications. The segments were fixed in 4% paraformaldehyde in 0.1 M PBS (4 mM sodium phosphate, pH 7.4, and 200 mM NaCl) for 12 hr at 4ЊC.…”