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Wat zijn ggo's? Wat is hun impact op milieu, mens en maatschappij? Waarom is het debat zo gepolariseerd? Ggo's worden steeds belangrijker voor onze voedselproductie en hun bijdrage aan een efficiëntere landbouw wordt volop bestudeerd. Maar ook het maatschappelijk debat omtrent ggo's woedt volop en lokt hevige reacties uit. Dit boek biedt u een alomvattend beeld van de kwestie, toegankelijk gebracht door experts uit verschillende disciplines.

Genetische manipulatie --- Crops --- Genetic engineering --- Food --- Biotechnology --- Genetica --- Landbouw --- 631.521 --- 631.521 Genetisch gemodificeerde organismen (GGO). Transgene organismen --- Genetisch gemodificeerde organismen (GGO). Transgene organismen

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The SnRK1 (SNF1-related kinase1) protein kinase, the ortholog of the yeast SNF1 (Sucrose non-fermenting1) kinase and animal AMP-activated kinase (AMPK), acts as a key metabolic sensor in plants. It integrates both endogenous developmental cues and diverse environmental stress conditions, that affect photosynthesis, respiration, or carbon allocation, triggering a coordinated response to ensure the maintenance of cellular energy homeostasis and survival. This involves metabolic and transcriptional reprogramming, also affecting plant growth. However, how exactly SnRK1 is activated upon increased energy demand or decreasing energy supply and how this affects growth and development is still largely unknow. To address these questions, we first developed and optimised a novel reporter to assess in vivo SnRK1 activity. The reporter is based on a peptide sequence of the well-known AMPK target Acetyl CoA Carboxylase 1 (ACC1) with S79 phosphorylation site, that is fused to an enhanced Green Fluorescent Protein (eGFP) tag (for subcellular localisation) and a double hemagglutinin (HA) tag (for protein expression analysis). The phosphorylation status of the reporter is evaluated using a commercially available phospho-ACC antibody. This construct can be used to study SnRK1 activity in vivo in isolated leaf mesophyll protoplasts or in intact Arabidopsis reporter plants constitutively expressing the reporter. After heterologous expression in bacteria and purification, it can also be used for in vitro kinase assays. Specific and conserved phosphorylation of the reporter was also confirmed in yeast using the S. cerevisiae snf1 mutant. Trehalose-6-phosphate (T6P), an intermediate of plant trehalose biosynthesis and an important novel growth regulator, was suggested to repress SnRK1 activity in young tissues under optimal growth conditions (Zhang et al., 2009). A major objective of this work is to study the molecular mechanisms involved. Using cellular (transient expression) and in vitro assays (using the ACC reporter), we confirmed that T6P regulates SnRK1 especially in younger tissues, and that T6P is not a direct regulator of the heterotrimeric SnRK1 complex, but requires an additional intermediary protein. We then explored the possible conserved regulation of yeast SNF1 by T6P, which would enable the use of powerful yeast genetics to elucidate the underlying molecular mechanisms in more detail. T6P regulation of SNF1 was suggested by the observation that the additional mutation of Snf1 in the tps1 mutant rescued the growth defect of this mutant on rapidly fermentable carbon sources. However, SNF1 T-loop phosphorylation, ACC reporter phosphorylation, and Mig1 (SNF1 target transcription factor) localisation in the WT and tps1 mutant indicated that T6P does not regulate SNF1 activity. Further investigation of gene expression, protein stability and activity of two SNF1 regulated enzymes fructose-1,6-bisphosphatase (encoded by FBP1) and phosphoenolpyruvate carboxykinase (PCK1), revealed a role for T6P in the regulation of gluconeogenesis, downstream and independent of SNF1 activity. The involvement of two SNF1-regulated transcription factors, Cat8 and Sip4, was assessed, but neither transcription factor seems to be involved in mediating the T6P regulation.The role of SnRK1 signalling in growth and development was investigated by gene expression profiling of growing leaves. Previous analyses in Arabidopsis rosette leaf series identified a remarkable peak in the expression of DIN6 (DARK-INDUCIBLE 6), a well-characterized SnRK1 target, suggesting a SnRK1-mediated metabolic and/or developmental transition, coinciding with the growth of the petiole and reduced sugar accumulation. Early leaf development involves a transition from cell division to cell expansion. Using the mesophyll protoplast assay, we found that SnRK1 can repress cyclin (CYCD3;1 and CYCB1;1) promoter activity, providing a direct link with growth control. Interestingly, transient expression of regulatory components in fully differentiated mesophyll leaf cells system can be used as a tool to study cell cycle regulatory machinery. However, the observed peak in DIN6 expression in developing leaves occurs after the transition from cell division to cell expansion. It also does not correspond to the juvenile-adult transition in larger rosettes. The expression of genes involved in sink and source activities indicate that the peak in DIN6 expression (and thus SnRK1 activity) might rather be associated with the transition from sink (net sucrose importing) to source (active photosynthesis and net sucrose exporting) activity, although in cellular assays overexpression of the SnRK1 catalytic subunit KIN10 mainly induced genes involved in sink activities.Finally, given its vital role in plant growth and development, further analyses of SnRK1 function are highly needed and would greatly benefit from specific direct small molecule SnRK1 activators and inhibitors. We therefore optimised the protoplast transient expression system for high throughput compound screening. New luciferase reporter constructs, using the deep sea shrimp nanoluciferase, were made resulting in an increased bioluminescence signal and simplification of the protocol to a one step process and optimal protoplast concentrations were determined.

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