We describe for distylous Turnera subulata a polygalacturonase specific to short-styled plants that is localized to the style transmitting tissue (the tissue through which pollen tubes grow). The polygalacturonase gene is linked to and may be upregulated by the S allele of the distyly locus. Because of its tissue-specific location, the polygalacturonase may be involved in the self-incompatibility response, acting in a complementary or antagonistic manner, or possibly in signalling downstream events. A pollen-specific polygalacturonase was also identified and may be a member of a small multigene family of pollen polygalacturonases. The role, if any, played by the pollen polygalacturonase in distyly, is presently unknown.
Background information. Within the group of lysosomal storage diseases, NPC1 [NPC (Niemann-Pick type C) 1] disease is a lipidosis characterized by excessive accumulation of free cholesterol as well as gangliosides, glycosphingolipids and fatty acids in the late E/L (endosomal/lysosomal) system (Chen et al., 2005) due to a defect in late endosome lipid egress. We have previously demonstrated that expression of the small GTPase Rab9 in NPC1 cells can rescue the lipid transport block phenotype (Walter et al., 2003), albeit by an undefined mechanism.Results. To investigate further the mechanism by which Rab9 facilitates lipid movement from late endosomes we sought to identify novel Rab9 binding/interacting proteins. In the present study, we report that Rab9 interacts with the intermediate filament phosphoprotein vimentin and this interaction is altered by lipid accumulation in late endosomes, which results in inhibition of PKC (protein kinase C) and hypophosphorylation of vimentin, leading to late endosome dysfunction. Intermediate filament hypophosphorylation, aggregation and entrapment of Rab9 ultimately leads to transport defects and inhibition of lipid egress from late endosomes.Conclusions. These results reveal a previously unappreciated interaction between Rab proteins and intermediate filaments in regulating intracellular lipid transport.
Abstract:We investigate sites of incompatibility for five Turnera L. species and for Piriqueta caroliniana (Walt.) Urban using aniline blue staining of pollen tubes and fluorescence microscopy. We show that sites of incompatibility occur in the stigma and upper regions of the style. There is an asymmetry between the morphs where, following selfing, pollen tubes tend to grow further into long-styled compared to short-styled plants, although the difference is not particularly marked. We demonstrate for the first time a qualitative difference in the appearance of pollen tubes. Upon self-pollination, pollen tubes in short-styled plants do not produce callose plugs, while all other pollinations result in callose plug formation. This could indicate that there is a difference in the mechanisms of incompatibility between the morphs. Exploiting crosses between two populations of Turnera scabra Millsp. that differ in flower size, we found no support for the hypothesis that incompatibility of long-styled plants is the result of insufficient nutrient reserves in pollen. Bud-pollinations provided limited support for the hypothesis that a short-specific style protein is involved in incompatibility. Crosses between distylous and homostylous species provided support for a recombinant origin for homostyly for four species, while deviations from this expectation were observed in four instances.Key words: distyly, homostyly, self-incompatibility, callose, pollen tubes, Turnera.Résumé : Les auteurs ont étudié les sites d'incompatibilité chez cinq espèces de Turnera L. et chez le Piriqueta caroliniana (Walt.) Urban, en utilisant la microscopie en fluorescense après avoir coloré les tubes polliniques au bleu d'aniline. On montre qu'il y a des sites d'incompatibilité dans les stigmates et dans les régions supérieures du style. Il y a asymétrie entre les morphes où, suite à l'autofécondation, les tubes polliniques ont tendance à pousser plus loin dans les styles longs que dans les styles courts, bien que la différence ne soit pas très marquée. On démontre pour la première fois une différence qualitative dans l'apparence des tubes polliniques. Suite à l'auto-pollinisation, chez les plantes à styles courts, les tubes polliniques ne produisent pas de bouchon de callose, alors que dans toutes les autres pollinisations il y formation de bouchons de callose. Ceci suggère une différence dans les mécanismes d'incompatibilité entre les morphes. En exploitant les croisements entre deux populations du Turnera scabra Millsp., qui diffèrent selon la dimension de leurs fleurs, les auteurs n'ont trouvé aucune preuve supportant l'hypothèse que l'incompatibilité chez les plantes à styles longs résulterait de réserves nutritives insuffisantes dans le pollen. Les pollinisations des bourgeons n'offrent qu'un support limité à l'idée qu'une protéine spécifique aux fleurs à style court, serait impliquée dans l'incompatibilité. Les croisements entre les espèces distyles et homostyles supportent l'idée de l'origine recombinatoire pour l'homostylie chez quatre ...
Extraction of DNA from plant tissue is often problematic, as many plants contain high levels of secondary metabolites that can interfere with downstream applications, such as the PCR. Removal of these secondary metabolites usually requires further purification of the DNA using organic solvents or other toxic substances. In this study, we have compared two methods of DNA purification: the cetyltrimethylammonium bromide (CTAB) method that uses the ionic detergent hexadecyltrimethylammonium bromide and chloroform-isoamyl alcohol and the Edwards method that uses the anionic detergent SDS and isopropyl alcohol. Our results show that the Edwards method works better than the CTAB method for extracting DNA from tissues of Petunia hybrida. For six of the eight tissues, the Edwards method yielded more DNA than the CTAB method. In four of the tissues, this difference was statistically significant, and the Edwards method yielded 27-80% more DNA than the CTAB method. Among the different tissues tested, we found that buds, 4 days before anthesis, had the highest DNA concentrations and that buds and reproductive tissue, in general, yielded higher DNA concentrations than other tissues. In addition, DNA extracted using the Edwards method was more consistently PCR-amplified than that of CTAB-extracted DNA. Based on these results, we recommend using the Edwards method to extract DNA from plant tissues and to use buds and reproductive structures for highest DNA yields.
To explore the genetic architecture of distyly in Turnera spp., we determined the inheritance and compatibility behaviour of two spontaneous homostyled mutants. A long-homostyled mutant shoot arose on an otherwise short-styled plant that was an artificial hybrid (Turnera subulata  T. krapovickasii) between two diploid distylous species. The mutation appears to be an allele, S H , of the distyly locus with the dominance relationships, S4SH 4s, where S confers the short-styled phenotype, S H confers homostyly in S H S H and S H s genotypes, and ss genotypes are long-styled. Aberrant segregation ratios were observed among some crosses and might be the result of pollen competition. Compatibility relationships are consistent with the hypothesis that a gene complex determines distyly. Infrequently, revertant short-styled flowers have appeared on cuttings of the T. subulata  T. krapovickasii mutant and on occasion, short-styled progeny have appeared in crosses where none were expected. A second mutant homostyle was discovered in autotetraploid T. scabra. The mutation is inherited as above, however, tetrasomic inheritance occurs at the locus. This homostyled mutant carries two copies of the S H allele and has the duplex genotype S H S H ss. Compatibility relationships were as observed above. The occurrence of homostyled mutants is consistent with the hypothesis that a linked gene complex underlies the inheritance of distyly in Turnera but we cannot discount the hypothesis that an allelic series is responsible.
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