Project description:Inflammatory Bowel Disease (IBD) is a term describing a collection of conditions characterised by chronic inflammatory disorder of the gastrointestinal tract involving an inappropriate immune response to commensal microorganisms in a genetically susceptible host. Four kiwifruit extracts, aqueous and ethyl acetate extracts of gold kiwifruit (Actinidia chinensis) or green kiwifruit (A. deliciosa), have previously demonstrated anti-inflammatory activity using in vitro models of IBD. This study examined whether these kiwifruit extracts had immune modulating effects in vivo against inflammatory processes known to be increased in patients with IBD. KFEs were used as a dietary intervention in Il10-/- mice (an in vivo model of IBD) and the C57BL/6J background strain in a 3 x 2 factorial design. While all Il10-/- mice developed significant colonic inflammation compared to the C57BL/6J mice, this was not affected by the inclusion of KFE in the diet. Whole genome gene and protein expression level profiling indicated that KFEs influenced immune signalling pathways and metabolic processes within the colonic tissue; however, the effects were subtle. In particular, adaptive immune pathways were reduced by three out of four kiwifruit extracts, with greater reduction seen in the C57BL/6J mice. This suggests that while immune-modulating activity was present in vivo, KFEs did not reduce inflammatory processes relevant to IBD. Experimental design. Two experiments were conducted, one using extracts from gold kiwifruit and one using extracts from green kiwifruit. Within each experiment, both Il10-/- and C57 mice were randomly divided into three diet treatment groups, to ive the following 12 treatments: 1) Gold Kiwifruit Experiment, C57BL/6J mice, Control diet (AIN-76A), 2) Gold Kiwifruit Experiment, C57BL/6J mice, Aqueous Kiwifruit Extract Supplemented Diet (AIN-76A + 5% Aqueous KFE) 3) Gold Kiwifruit Experiment, C57BL/6J mice, Ethyl Acetate Kiwifruit Extract Supplemented Diet (AIN-76A + 0.11% Ethyl Acetate KFE) 4) Gold Kiwifruit Experiment, Il10-/- mice, Control diet (AIN-76A), 5) Gold Kiwifruit Experiment, Il10-/- mice, Aqueous Kiwifruit Extract Supplemented Diet (AIN-76A + 5% Aqueous KFE) 6) Gold Kiwifruit Experiment, Il10-/- mice, Ethyl Acetate Kiwifruit Extract Supplemented Diet (AIN-76A + 0.11% Ethyl Acetate KFE) 7) Green Kiwifruit Experiment, C57BL/6J mice, Control diet (AIN-76A), 8) Green Kiwifruit Experiment, C57BL/6J mice, Aqueous Kiwifruit Extract Supplemented Diet (AIN-76A + 5% Aqueous KFE) 9) Green Kiwifruit Experiment, C57BL/6J mice, Ethyl Acetate Kiwifruit Extract Supplemented Diet (AIN-76A + 0.11% Ethyl Acetate KFE) 10) Green Kiwifruit Experiment, Il10-/- mice, Control diet (AIN-76A), 11) Green Kiwifruit Experiment, Il10-/- mice, Aqueous Kiwifruit Extract Supplemented Diet (AIN-76A + 5% Aqueous KFE) 12) Green Kiwifruit Experiment, Il10-/- mice, Ethyl Acetate Kiwifruit Extract Supplemented Diet (AIN-76A + 0.11% Ethyl Acetate KFE) Six biological replicates were analysed from each treatment group, except for groups 3 and 7 where five replicates were analysed due to array quality issues with the sixth replicate. Each replicate contained mRNA from one mouse. A reference design was used, where each slide was hybridised with mRNA from one sample (green channel) and mRNA from a common reference pool (red channel).

Project description:Purpose: The goals of this study was to provide genome-wide data to investigate the molecular mechanism of the interaction between ABA and ethylene during tomato fruit ripening Methods: Regarding the fruits sampled at the 9th day as a well-characterized stage, we used RNA-seq to conducted a comparative analysis of exogenous ABA and NDGA effects on all components involved in biosynthesis and signaling of ethylene and ABA.The identified crucial genes in response to ABA were further performed a ripening time-course analysis by RT-PCR. In addition, we also detected how ethylene affected ABA action at the onset of ripening by treating the fruits with 1-MCP immediately after ABA application. Results: Our study not only illustrated how ABA regulated itself at the transcription level, but also elucidated that ABA can facilitate ethylene production and response by regulating some crucial genes such as LeACS4, LeACO1 and LeETR6 etc. In addition, investigation on the fruits treated with 1-MCP immediately after ABA exposure revealed that ethylene might be essential for the induction of ABA biosynthesis and signaling at the onset of fruit ripening. Furthermore, some specific transcription factors (TFs) known as regulators of ethylene synthesis and sensibility (e.g. MADS-RIN, TAGL1, CNR and NOR etc.) were also observed to be ABA responsive, which implied that ABA influenced ethylene action possibly through the regulation of these TFs expression. Conclusions: Our data suggested that ABA may act as an upstream regulator to modulate ethylene synthesis and signal transduction, which consequently influenced the ripening process. This comprehensive survey not only demonstrated how ABA regulated itself at the molecular level, but also indicated that ABA had a positive impact on ethylene production and action by regulating key genes such as LeACS2, LeACS4, LeACO1 and LeETR6. Besides, our results also revealed that ethylene might be of great importance to induce ABA accumulation and response at the onset of ripening. Moreover, many ripening related TFs, such as MADS-RIN, TAGL1, CNR and NOR, were observed to be affected by ABA, implying that a TF-mediated manner may be involved in the interaction between ABA and ethylene. This study extends our understanding of the mechanism that how ABA-triggered tomato fruit ripening, and illustrates the complex mechanism of reciprocity between ABA and ethylene at the transcription level.

Project description:Tomato fruit ripening is under the control of ethylene as well as a group of ethylene-independent transcription factors, including NON-RIPENING (NOR) and RIPENING INHIBITOR (RIN). During ripening, the linear carotene lycopene accumulates at the expense of cyclic carotenoids. Fruit-specific overexpression of LYCOPENE β-CYCLASE (LCYb) under the control of the PHYTOENE DESATURASE (PDS) promoter resulted in increased levels of β-carotene and ABA and in decreased ethylene levels. Genes regulated by ABA, or involved in its synthesis and signaling, were overexpressed, while those associated with ethylene and cell wall remodeling were repressed. In agreement with the transcriptional data, LCYb-overexpressing fruits exhibited increased density of cell wall material containing linear, under-methylated pectins and displayed an array of additional ripening phenotypes, including delayed softening, increased turgor, enhanced shelf life and a thicker cuticle with a higher content of cutin monomers and triterpenoids. The levels of several primary metabolites and phenylpropanoids also changed in the transgenics, which could be attributed to delayed fruit ripening and to ABA respectively. Network correlation analysis suggests that ABA, acting through NOR and RIN, is responsible for many of the above phenotypes. These data reinforce suggestions that ABA plays an important role in tomato fruit ripening and provide clues that fruit b-carotene, acting as a precursor for ABA, actively participates in controlling the ripening process rather than merely being an output thereof. Overexpression of a LCYb gene from Arabidopsis under the control of the ripening-associated PDS promoter leads to ripe tomato fruits accumulating high β-carotene levels. Using several independent transgenic lines, we conducted a system-wide study of the effect of increased β-carotene levels on tomato fruit ripening and shelf life. Our data suggest that β-carotene, acting through ABA, is involved in a regulatory loop within the network controlling tomato fruit ripening.

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