Methoder og protokoller

Routine chemical analysis

N and related metabolites:

One of the main research topics in the laboratory is the analysis of N and it’s metabolites (nitrate, ammonium and amino acids) in plants and soil.

• Nitrates are analysed by flow injection analysis (FIA) on a Lachat instrument.
• Ammonium is now analysed by fluorescence spectrometry (96 well microplate technique)
• Amino acids are analysed by reverse phase HPLC using derivatisation by AccQ-Tag and fluorescence detection.
• Stable isotope enrichment (15N) in all species may be isolated and determined by CF-IRMS as described below.

Elemental analysis:

• Total N, C and their stable isotopes (15N/14N and 13C/12C) are usually analysed by Continuous-Flow stable Isotope Ratio Mass Spectrometry (CF IRMS). We have an Europa Scientific ANCA-SL elemental analyser coupled to a 20-20 Mass Spectrometer.
• Traditional single element analyses (P, K, Mg, Ca) may be carried out by AAS and GF-AAS equipment also available in the laboratory.
• Multi-elemental analysis and sub-ppb analysis of essential plant nutrients are usually performed by Inductively Coupled Plasma Mass Spectrometry (ICP-MS). For high-throughput profiling of multiple inorganic elements and their speciation in various plant tissues we have an Agillent 7500c ICP-MS equipped with octopole collision/reaction cell and hyphenated with an HPLC The ICP-MS is housed in newly constructed ISO-certified clean-room facility and is equipped with an octopole reaction cell enabling accurate measurements at the ppt-level of even problematic trace elements such as Cr, Fe and Se in small quantities of tissue
• The laboratory is fully equipped with certified clean-room facilities and various equipment to produce ultra-pure water and acids. In addition both open and closed non-metallic systems for digestion of samples are available.

Other analyses

• Plant fibre content according to the van Soest method (solubles, hemicellulose, cellulose and lignin) may be carried out using an Ankom 220 Fibre Analyser
• Chlorofyll in plant leaves is carried out by standard spectrophotometry

 

Basic molecular biology techniques:

Heterologous expression: The yeast Saccharomyces cerevisiae has proven to be a powerful tool for the functional characterization of plant transporters. Nutrient translocation in yeast is often catalyzed by transport proteins with homology to plant transporters. Mutants deficient in the respective genes of such specific transporters often show a growth defect, under certain environmental conditions and can thus be used to clone and functionally characterize the plant homologue – a method known as functional and homologous complementation.

 

Yeast can, however, also be used to clone members of non-homologous proteins with analog function. We have further extended the use of yeast for the cloning soluble proteins. Thus, yeast, albeit of its long tradition as a tool in plant molecular biology is still one of the most convenient and up to date tools for the cloning and characterization of plant proteins.

 

We have ongoing co-operations with excellent facilities for heterologous expression in Xenopus oocytes. Altered expression levels of particular genes in plants is a means to elucidate protein function. On the routine basis, we generate and analyse sense or anti-sense over-expressing plants to characterize the function of particular proteins in plant physiology.

 

In addition T-DNA knockout plants and plant expressing interfering RNA (RNAi) are used to completely delete single ORF or even subfamilies of homologous proteins.