Bruce A. McClure

Action of the Style Product of the Self-Incompatibility Gene of Nicotiana alata (S-RNase) on in Vitro-Grown Pollen Tubes.

The products of the S-locus expressed in female tissues of Nicotiana alata are ribonucleases (S-RNases). The arrest of growth of incompatible pollen tubes in styles may result from entry of the S-RNase into the pollen tube and degradation of pollen tube RNA. We investigated the action of isolated S-RNases on pollen tubes grown in vitro and found that S-RNase is taken up by the pollen without substantial alteration. The S-RNases inhibit incorporation of exogenously added radioactive amino acids into protein by the germinated pollen. The S-RNases also inhibit in vitro translation of pollen tube RNA in a wheat germ cell-free extract. We found no evidence for a specific mRNA substrate for the S-RNases, which implies that if RNase activity is involved in the control of self-incompatibility, allelic specificity is more likely to depend on the selective uptake of S-RNases into pollen tubes or their selective activation or inactivation by pollen factors, rather than cleavage of a specific substrate. Heat treating S2-RNase largely destroys its RNase activity but increases its inhibitory effect on in vitro pollen tube growth. This effect is not due to an increased uptake of S2-RNase by the pollen but is associated with a greatly enhanced accumulation of S2-RNase on the outer surface of the pollen grains.

Tissue print hybridization. A simple technique for detecting organ- and tissue-specific gene expression

We have used a new technique, which we call tissue print hybridization, to monitor organ- and tissue-specific expression of auxin-induced RNAs in soybean (Glycine max cv. Wayne) seedlings. This technique is modified from that originally published by Cassab and Varner (J Cell Biol 105: 2581–2588, 1987) for the localization of extensin protein in soybean seed using an antibody probe. We extended this original tissue print procedure by utilizing35S-labeled antisense RNAs for localization of specific RNAs immobilized on nylon membranes. We also employed modifications to improve the resolution of the autoradiographic images. We have used this technique to demonstrate the tissue-specific expression of auxin-regulated genes in elongating hypocotyl regions of etiolated soybean seedlings and the rapid turn-over of RNAs encoded by these genes during gravistimulation.

AMPLIFICATION OF THE POLYMORPHIC 5.8S rRNA GENE FROM SELECTED AUSTRALIAN GIGARTINALEAN SPECIES (RHODOPHYTA) BY POLYMERASE CHAIN REACTION1

We have initiated comparative studies of ribosomal RNA (rRNA) gene structure to explore its potential to provide taxonomically useful data within the large red algal order Gigartinales. In southern Australia, this group is extremely diverse and includes large numbers of endemic taxa, many of potential economic importance. The 5.8S rRNA gene occurs in the middle region of the ribosomal DNA (rDNA) cistron and is flanked by two internal transcribed spacers (ITSs). These spacers contain regions of DNA, which are highly consented at the generic level and above, interspersed with highly divergent sequences. The 5.8S and associated ITS s of 11 species of Gigartinales (including five species of the largest Australian endemic marine algal genus, Mychodea), plus five taxa belonging to other orders, were amplified by the polymerase chain reaction. The size of the 5.8S rDNA and its flanking ITSs varied not only within and between genera, but also at the species level. However, this rDNA sequence appears to be relatively constant within populations find may be useful as a populational marker.

Expression of a Self-Incompatibility Glycoprotein (S2-Ribonuclease) from Nicotiana alata in Transgenic Nicotiana tabacum.

In Nicotiana alata, self-incompatibility is controlled by a single locus, designated the S-locus, with multiple alleles. Stylar products of these alleles are ribonucleases that are secreted mainly in the transmitting tract tissues. N. tabacum plants were transformed with constructs containing the S2-cDNA and genomic S2-sequences from N. alata that were linked to the cauliflower mosaic virus 35S promoter. Unlike other genes controlled by this promoter, the genes were expressed most highly in mature floral organs. This pattern of expression was observed at both the protein and RNA levels. The S2-glycoprotein was detected in the stylar transmitting tract tissues of the transgenic plants. The transgene product was secreted, had ribonuclease activity, and was glycosylated with the correct number of glycan chains. However, the maximum level of S2-glycoprotein in styles of the transgenic plants was approximately 100-fold lower than that found in N. alata styles carrying the S2-allele. Perhaps because of this lower protein level, the plants showed no changes in the incompatibility phenotype.

S-locus products in Nicotiana alata pistils are subject to organ-specific post-transcriptional processing but not post-translational processing

The expression of genes encoding self-incompatibility ribonucleases (S-RNases) in Nicotiana alata were examined at both protein and RNA level for organ specificity. S-RNases recovered from stigmas and styles were indistinguishable by SDS-PAGE, chromatographic behaviour and RNase specific activity. The pistil S transcripts are heterogenous in size, the stigma transcript being shorter and more heterogenous than the transcripts in the style and ovary. RNase H analysis shows that this organ-specific difference is mainly in the length of the polyadenylate tail. By sequence analysis of cloned cDNAs we show that the transcript present in the stigma is derived from the same gene as the transcript in the style.

Molecular Aspects of Self-incompatibility in the Solanaceae

Many species of flowering plants have evolved genetically controlled mechanisms to prevent inbreeding. Fertilization in flowering plants involves a complex series of interactions between the haploid pollen which contains the male gametes, and the diploid tissues of the female pistil. Flowers are often hermaphroditic bearing the male and female organs in close proximity, so that the effectiveness of these mechanisms in preventing self-fertilization and promoting outcrossing is particularly important. Self-incompatibility (SI) is one of the most widespread mechanisms for preventing self-fertilization. It is the inability of seed plants to produce viable seeds after self-pollination. Study of the mechanism of self-incompatibility is not only of interest in relation to pollination but also as a model system for understanding cell-cell recognition in higher plants.

Self-Incompatibility as a Model for Cell-Cell Recognition in Flowering Plants

Chemical signals which are secreted from one cell type are known to recognise and exert an effect on a specific target cell or cells. In animal systems, recognition of this type is usually achieved by the signal molecule, for example a hormone, binding to a specific receptor on the surface of the target cell. The signal molecule may then be internalised by receptor mediated endocytosis, or alternatively, receptor binding may initiate a second messenger response resulting in an intracellular effect. In plant systems cell-cell interactions are not as clearly understood. Plant cells secrete hormone like substances such as auxins, cytokinins and gibberellins. Receptor mediated endocytosis has recently been demonstrated in cultured soybean cells (Horn et al., 1989) and cyclic AMP, protein kinases, calmodulin and coated pits, which are involved in animal signalling systems, have all been identified in plants. It is therefore possible that in spite of the differences between plant and animal cells, the most important being the presence of a cell wall, analogous cellular recognition systems may operate. Indeed, components of the cell wall, known as “oligosaccharins” have themselves been implicated as hormones (Eberhard et al., 1989).

[54] – Tissue-Print Hybridization for Detecting RNA Directly

Publisher Summary nTissue printing is a simple method for detecting macromolecules blotted directly from the surfaces of severed organs onto nylon or nitrocellulose membranes. The blotting procedure produces an image of the cut surface of the tissues on the membrane. Macromolecules such as proteins, complex carbohydrates, and nucleic acids are fixed to the membrane. The retention of nucleic acids on the membrane allows the detection of RNAs by hybridization with either DNA or antisense RNA probes. For tissue printing, a dry nylon membrane is placed over a single layer of dry whatman paper or some other absorbent paper. Organs or organ sections are prepared for printing onto membranes by sectioning through the organ with a single- or double-edged razor blade. The freshly cut surfaces are pressed immediately to the nylon membrane or lightly blotted with kimwipes prior to blotting to the membrane. Tissue printing is performed by using firm pressure with the index finger above the sectioned organ for 30–120 sec. The quality of the tissue prints is evaluated by examining the printed nylon membrane under a UV light source. Under UV light, it is possible to observe whether any organ sections were crushed or distorted during blotting. It is found that the large organs of firm consistency such as coytledons, stems, and petioles are much easier to tissue print than small or less firm organs such as roots, leaves, or floral parts.

Molecular Genetics of Self-incompatibility in Nicotiana alata

Self-incompatibility is a genetically controlled mechanism which prevents inbreeding in plants (de Nettancourt, 1977). In many, but not all cases, it is controlled by a multi-allelic, single gene, the S-gene. The system operates to enhance outcrossing and to ensure that a plant is fertilized by a genetically distinct individual of the same species. There are two major types of self-incompatibility. The most widespread is gametophytic self-incompatibility which involves interaction of a product of the haploid genome of the male gametophyte (carried within the pollen grain) and a product of the diploid genome of the female tissue of the sporophyte, the pistil. In incompatible matings, as is the case when the S-allele carried by the haploid pollen matches either of the S-alleles present in the diploid style, pollen tube growth is arrested within the transmitting tract (Figure 1).