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Bryan Penning
*Supported bythe the NSF Supported by NSF Plant Genome Research and Plant Genome Research REU Programs and REU Programs
Overview
We are establishing infrared spectrotypes in cell wall mutants. Spectrotypes are spectroscopic phenotypes, i.e. the spectral differences between mutant and wild type populations. This tutorial will:
Give some background information on how we prepare and collect data on mutants (See Techniques VII at: http://cellwall.genomics.purdue.edu/techniques/7.html for more details) Show how we analyze the data collected by Principal Components Analysis (PCA) and digital subtraction Give some example spectral peaks that indicate differences in cell wall composition between mutants and wildtype
All plants are grown and cell walls isolated under the same conditions and processed simultaneously for internal consistency of mutant and wild type comparisons (see Techniques VII for greater detail: http://cellwall.genomics.purdue.edu/techniques/7.html)
Plant preparation
Wild type plants are always prepared with mutant plants Plants are all grown on one-half strength MS salts with 1% sucrose and 0.8% agar in the light for 12 days Plants are all transferred to the dark for 2 days to lower starch content (a contaminating feature in IR spectrum) Plant tissue is crushed in liquid nitrogen, and non cell wall components, such as proteins, are extracted using an SDS-Tris buffer Cell walls are isolated by homogenization in a Geno-Grinder (SPEX Certi-Prep), collected on a nylon mesh and washed with water and ethanol Cell walls are resuspended in distilled water and spotted on a gold-plated slide (EZ-Spot, Spectra-Tech) for spectral acquisition in an FTIR spectrometer (Thermo-Electron, Madison, WI )
Fig. A: We collect many spectra (about 40) with a computer driven stage and Omnic software (Thermo Electron), saving them as grouped data (*.spa) Fig. B: We convert group data to individual *.jdx files in Omnic
Supported by the NSF Plant Genome Research and REU Programs
* Kemsley. 1998.
In Win-Das we:
Digital subtraction
To perform a digital subtraction, average the mutant and wild-type spectra (previous slide) and copy over the spectra values (left column of spectra files) Subtract mutant from wild type and plot versus wavenumber (cm-1) Look for peaks (differences in cell wall components)
However, these peak assignments are based on isolated polysaccharides and peaks may shift depending on molecular interactions and environment within the cell wall The more peaks that can be assigned to a particular polymer, the more likely that component differs between mutant and wild type cell walls
* Kaurkov, Capek, Sasinkov, Wellner, and Ebringerov. 2000. FT-IR study of plant cell wall model compounds: pectic polysaccharides and hemicelluloses. Carbohydrate Polymers 43:195- 203
Supported by the NSF Plant Genome Research and REU Programs