FRET-FLIM applications in plant systems

A hallmark of cellular processes is the spatio-temporally regulated interplay of biochemical components. Assessing spatial information of molecular interactions within living cells is difficult using traditional biochemical methods. Developments in green fluorescent protein technology in combination with advances in fluorescence microscopy have revolutionised this field of research by providing the genetic tools to investigate the spatio-temporal dynamics of biomolecules in live cells. In particular, fluorescence lifetime imaging microscopy (FLIM) has become an inevitable technique for spatially resolving cellular processes and physical interactions of cellular components in real time based on the detection of Förster resonance energy transfer (FRET). In this review, we provide a theoretical background of FLIM as well as FRET-FLIM analysis. Furthermore, we show two cases in which advanced microscopy applications revealed many new insights of cellular processes in living plant cells as well as in whole plants.     

Assessment of dietary intake: NuGO symposium report

Advances in genomics science and associated bioinformatics and technology mean that excellent tools are available for characterising human genotypes. At the same time, approaches for characterising individual phenotypes are developing rapidly. In contrast, there has been much less investment in novel methodology for measuring dietary exposures so that there is now a significant gap in the toolkit for those investigating how diet interacts with genotype to determine phenotype. This symposium reviewed the strengths and limitations of current tools used in assessment of dietary intake and the potential to improve these tools through, for example, the use of statistical techniques that combine information from different sources (such as modelling and calibration methods) to ameliorate measurement error and to provide validity checks. Speakers examined the use of approaches based on technologies such as mobile 'phones, digital cameras and Web-based systems which offer the potential for more acceptable (for study participants) and less laborious (for researchers and participants) routes to more robust data collection. In addition, the application of omics, especially metabolomics, tools to biofluids to identify new biomarkers of intake offers great potential to provide objective measures of food consumption with the advantage that data may be collected in forms that can be integrated readily with other high throughput (nutrigenomic) technologies.     

Mouse strain differences in autonomic responses to stress

In humans, anxiety disorders are often accompanied by an overactive autonomic nervous system, reflected in increased body temperature (BT) and heart rate (HR). In rodents, comparable effects are found after exposure to stress. These autonomic parameters can give important information on stress and anxiety responses in mice. In the present experiments, stress reactivity of three frequently used mouse strains [129 Sv/Ev, Swiss Webster (SW) and C57 BL/6] was assessed using their autonomic stress responses. BT, HR and activity were telemetrically measured. Undisturbed circadian rhythms already showed clear differences between the mouse strains. Hereafter, autonomic responses to stressors with increasing intensity were measured. Strain differences were found in magnitude and duration of the stress responses, especially after high-intensity stressors. Generally, C57BL/6 mice showed the largest autonomic response, SW the lowest and the 129Sv/Ev the intermediate response. Interestingly, the observed ranking in autonomic stress response does not match the behavioral stress responsivity of these strains. Finally, sensitivity to the anxiolytic diazepam (0, 1, 2, 4 and 8 mg/kg) was tested using the stress-induced hyperthermia paradigm. Pharmacological sensitivity to diazepam differed between the strains with the 129Sv/Ev being most sensitive. These studies show that simultaneous measurement of behavioral and autonomic parameters under stressful conditions contributes considerably to a better interpretation of anxiety and stress levels in mice.     

Application of Petri net based analysis techniques to signal transduction pathways

Background  

Signal transduction pathways are usually modelled using classical quantitative methods, which are based on ordinary differential equations (ODEs). However, some difficulties are inherent in this approach. On the one hand, the kinetic parameters involved are often unknown and have to be estimated. With increasing size and complexity of signal transduction pathways, the estimation of missing kinetic data is not possible. On the other hand, ODEs based models do not support any explicit insights into possible (signal-) flows within the network. Moreover, a huge amount of qualitative data is available due to high-throughput techniques. In order to get information on the systems behaviour, qualitative analysis techniques have been developed. Applications of the known qualitative analysis methods concern mainly metabolic networks. Petri net theory provides a variety of established analysis techniques, which are also applicable to signal transduction models. In this context special properties have to be considered and new dedicated techniques have to be designed.     

Gdnf Haploinsufficiency Causes Hirschsprung-Like Intestinal Obstruction and Early-Onset Lethality in Mice

Hirschsprung disease (HSCR) is a common congenital disorder that results in intestinal obstruction and lethality, as a result of defective innervation of the gastrointestinal (GI) tract. Despite its congenital origin, the molecular etiology of HSCR remains elusive for >70% of patients. Although mutations in the c-RET receptor gene are frequently detected in patients with HSCR, mutations in the gene encoding its ligand (glial cell line-derived neurotrophic factor [GDNF]), are rarely found. In an effort to establish a possible link between human HSCR and mutations affecting the Gdnf locus, we studied a large population of mice heterozygous for a Gdnf null mutation. This Gdnf(+/-) mutant cohort recapitulates complex features characteristic of HSCR, including dominant inheritance, incomplete penetrance, and variable severity of symptoms. The lack of one functioning Gdnf allele causes a spectrum of defects in gastrointestinal motility and predisposes the mutant mice to HSCR-like phenotypes. As many as one in five Gdnf(+/-) mutant mice die shortly after birth. Using a transgenic marking strategy, we identified hypoganglionosis of the gastrointestinal tract as a developmental defect that renders the mutant mice susceptible to clinical symptoms of HSCR. Our findings offer a plausible way to link an array of seemingly disparate features characteristic of a complex disease to a much more narrowly defined genetic cause. These findings may have general implications for the genetic analysis of cause and effect in complex human diseases.     

On the evidence of a diffusion barrier in the outer cortex apoplast of cress-roots (Lepidium sativum), demonstrated by analytical electron microscopy

To mark the apoplastic pathway of ions in the root of the dicotyledonous plant Lepidium sativum we used the heavy element lanthanum, which can be identified by analytical electron microscopy (EELS and ESI). In the front root tip, the primary walls of all meristematic cells contained lanthanum. 10-15 mm behind the root apex, lanthanum was found in the cortex cell walls up to the endodermis, but not in the stele. 20-25 mm from the tip, lanthanum was accumulated in the radial cell walls of the hypodermis, which, however, is not a complete diffusion barrier for ions, so that traces of lanthanum also were found in the cortex cell walls up to the endodermis. This study provides evidence for the presence of two apolastic diffusion barriers in the region of highest water uptake in cress roots.     

Evidence for biological nature of the grape replant problem in California

A bioassay was developed to investigate causes of grape replant problems under controlled conditions. Soils were collected from methyl bromide-fumigated and non-fumigated plots at a site cleared from a 65-year-old grape vineyard (Vitis vinifera cv. Thompson seedless) at Parlier, CA. Subsamples of the non-fumigated soil were either left non-treated, subjected to autoclaving (twice 45 min), or heating at 40, 50, 60, 70, 80 or 90 °C for 30 min. Subsequently, the samples were placed in 120-mL pots, planted with rooted hardwood grape cuttings (V. vinifera, cv. Carignane) and placed in a greenhouse or growth chamber. Three months after transplanting, vines from non-treated or 40 °C-treated soil had lower shoot weights and densities of healthy lateral roots than vines from the other treatments. Pythium spp. were isolated from 45 to 55% of the plated root segments from vines grown in non-treated, or soil that had been heated at 40 or 50 °C but were not detected in roots from soil given other treatments. Egg masses of root-knot nematode, Meloidogyne spp., were produced on roots from non-treated or heated at 40 °C soil, but no egg masses were detected on roots of the other treatments. In another test with the same soils, remnant roots from non-fumigated or pre-plant methyl bromide-fumigated soil were extracted and amended to non-fumigated soil, soil from fumigated field plots, soil fumigated in a small container, or autoclaved potting mix. The transfer of old vine roots from non-fumigated field soil resulted in incidence of Pythium spp. on grape assay roots, but there was no measurable effect of the transfer on growth and health of the bioassay plant roots. The results of the bioassays indicate that grape replant problem at the California site had biological causes. The bioassay approach may aid in future determinations of the etiology of grape replant problems.