Overexpression of plum auxin receptor PslTIR1 in tomato alters plant growth, fruit development and fruit shelf-life characteristics.

BACKGROUND: TIR1-like proteins are F-box auxin receptors. Auxin binding to the F-box receptor proteins promotes the formation of SCF(TIR1) ubiquitin ligase complex that targets the auxin repressors, Aux/IAAs, for degradation via the ubiquitin/26S proteasome pathway. The release of auxin response factors (ARFs) from their Aux/IAA partners allows ARFs to mediate auxin-responsive changes in downstream gene transcription. In an attempt to understand the potential role of auxin during fruit development, a plum auxin receptor, PslTIR1, has previously been characterized at the cellular, biochemical and molecular levels, but the biological significance of this protein is still lacking. In the present study, tomato (Solanum lycopersicum) was used as a model to investigate the phenotypic and molecular changes associated with the overexpression of PslTIR1. RESULTS: The findings of the present study highlighted the critical role of PslTIR1 as positive regulator of auxin-signalling in coordinating the development of leaves and fruits. This was manifested by the entire leaf morphology of transgenic tomato plants compared to the wild-type compound leaf patterning. Moreover, transgenic plants produced parthenocarpic fruits, a characteristic property of auxin hypersensitivity. The autocatalytic ethylene production associated with the ripening of climacteric fruits was not significantly altered in transgenic tomato fruits. Nevertheless, the fruit shelf-life characteristics were affected by transgene presence, mainly through enhancing fruit softening rate. The short shelf-life of transgenic tomatoes was associated with dramatic upregulation of several genes encoding proteins involved in cell-wall degradation, which determine fruit softening and subsequent fruit shelf-life. CONCLUSIONS: The present study sheds light into the involvement of PslTIR1 in regulating leaf morphology, fruit development and fruit softening-associated ripening, but not autocatalytic ethylene production. The results demonstrate that auxin accelerates fruit softening independently of ethylene action and this is probably mediated through the upregulation of many cell-wall metabolism genes.

Toxicity of cholesterol oxidation products to Caco-2 and HepG2 cells: modulatory effects of alpha- and gamma-tocopherol.

Cholesterol can be oxidized to form a variety of cholesterol oxidation products also known as oxysterols. The aims of the present study were to compare the cytotoxic effects of four oxysterols, namely 25-hydroxycholesterol (25-OHC), 7beta-hydroxycholesterol (7beta-OHC), cholesterol-5beta,6beta-epoxide (beta-epox) and cholesterol-5alpha,6alpha-epoxide (alpha-epox), in two human cell culture models. Further, the ability of 10 and 100 micro m alpha- and gamma-tocopherol (alpha-TOC and gamma-TOC, respectively) to protect against oxysterol-induced cytotoxicity was also assessed. Human colonic adenocarcinoma Caco-2 and human hepatoma HepG2 cells were supplemented with increasing concentrations of 25-OHC, 7beta-OHC, beta-epox and alpha-epox (0-25 micro g ml(-1)) for 24, 48 or 96 h. Following 24-h and 48-h exposure, test media were replaced with normal growth media and the cells were maintained for 72 and 48 h, respectively. The 96-h exposure represented a constant challenge to the cells. Cytotoxicity was assessed using the neutral red uptake assay. The concentration of compound that inhibited cell viability by 50% (ic(50) value) was calculated. All four oxysterols investigated induced the greatest cytotoxic effects following 96 h of exposure. 25-Hydroxycholesterol exhibited the greatest cytotoxicity in both cell lines. Both beta-epox and alpha-epox were more toxic to HepG2 cells than to Caco-2 cells after the 48-h exposure. Pretreatment of cells with either alpha- or gamma-TOC did not protect against oxysterol-induced cytotoxicity. The caco-2 cells treated with the high concentration (100 micro m) of gamma-TOC were found to be more susceptible to oxysterol-induced toxicity under the conditions employed in this study.

Cell cycle-dependent repetitive Ca(2+ )waves induced by a cytosolic sperm extract in mature ascidian eggs mimic those observed at fertilization.

Sperm-triggered Ca(2+) oscillations occur throughout the animal kingdom. The mechanism sperm use to trigger Ca(2+) oscillations at fertilization has not been resolved in any egg. The temporal, spatial and regulatory characteristics of the Ca(2+) oscillations during fertilization in ascidians offer a unique advantage over other systems for determining the mechanism of fertilization. For example, sperm trigger two phases of Ca(2+) oscillations that are all waves in ascidians. The first of these Ca(2+) waves begins at the point of sperm-egg fusion while a second phase of Ca(2+) waves originates at a vegetal protrusion termed the contraction pole. In addition, cyclin B1-dependent kinase activity provides a form of positive feedback, maintaining the second phase of Ca(2+) waves during meiosis and thereby ensuring meiotic exit. We therefore prepared cytosolic ascidian sperm extracts or MonoQ-fractionated ascidian sperm extracts from this urochordate to investigate if a Ca(2+)-releasing sperm-borne factor was responsible for egg activation. Spatially, ascidian sperm extract induced repetitive Ca(2+) waves that mimicked the spatial pattern displayed during fertilization: all the second-phase Ca(2+) waves originated at a vegetal protrusion termed the contraction pole (thus mimicking fertilisation). We also demonstrated that ascidian sperm extract-induced Ca(2+) oscillations were maintained when CDK activity was elevated and MAP kinase activity was low, as found previously for sperm-triggered Ca(2+) oscillations. As would be predicted, large doses of ascidian sperm extract injected into prophase-stage oocytes, lacking CDK activity, failed to induce any Ca(2+) release even though they responded to microinjection of the Ca(2+)-releasing second messenger inositol 1,4,5-trisphosphate. Finally, since the Ca(2+)-releasing activity from Mono-Q fractionated ascidian sperm extract eluted predominantly as one fraction, this may imply that one factor is responsible for the Ca(2+)-releasing activity. These data support a model of egg activation whereby the sperm introduces a Ca(2+)-releasing cytosolic factor into the egg. We demonstrated that ascidian sperm contain a protein factor(s) that is regulated by the egg CDK activity and can trigger all the Ca(2+ )waves observed at fertilization with a spatial pattern that mimics those initiated by sperm.