Vibrational spectroscopy study of starch and cell wall polysaccharide structures. N Wellner Institute of Food Research Norwich, UK

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1 Vibrational spectroscopy study of starch and cell wall polysaccharide structures N Wellner Institute of Food Research Norwich, UK

2 Outline IR spectroscopy, normal modes and molecular structure Composition of plant tissue Functional imaging of film fracture Investigation of starch granules

3 IR spectra of polysaccharides Wheat starch Microcrystalline cellulose

4 IR spectra of polysaccharides

5 IR/Raman State of molecules described by Schrödinger Equation HY = EY Born-Oppenheimer approximation separates terms for electrons and nuclei, and also allows separation of the wave function into distinct components Oscillator potential (symmetric/asymmetric) and energy levels Interaction with light <y * m y > FTIR Absorption depends on dipole moment change Raman scattering depends on polarisability tensor (Source: Wikipedia)

6 IR spectra of polysaccharides Wheat starch Microcrystalline cellulose

7 Absorbance Interpreting the spectra OH stretch normal mode Structure modelled from Popov et al. Macromolecules 2009, 42, Wavenumber (cm -1 )

8 Absorbance Interpreting the spectra 940 cm -1 band Structure modelled from Popov et al. Macromolecules 2009, 42, Wavenumber (cm -1 )

9 Absorbance Structure and spectrum Methyl-a-Dglucopyranoside CH 2 OH O OH OH OCH 3 OH Methyl-a-Dmannopyranoside CH 2 OH O OH OH OH OCH ,800 1,600 1,400 1,200 Wavenumber (cm -1 ) 1,

10 Absorbance Mixtures spectra of plant materials 0.60 cellulose + hemicellulose lignin straw 0.30 protein starch maize kernel water CWM + starch carrot Wavenumber (cm -1 )

11 Carbohydrate metabolism in arabidopsis sucrose invertase glucose + fructose sucrose synthase UDPglucose + fructose cellulose synthase cellulose

12 Wavenumber ( cm -1 ) FT-IR imaging: spatially resolved chemical composition FTIR microscope Focal plane array detector with 128 x 128 elements Each 5 x 5 mm 2 element records a spectrum Spectra from regions or Images obtained by colouring pixels according to spectra information Y X

13 Changes in cell wall metabolism - sucrose synthase mutants Barratt et al., PNAS 2009, 106/31,

14 FTIR/Raman State of molecules described by Schrödinger Equation HY = EY Born-Oppenheimer approximation separates terms for electrons and nuclei, and also allows separation of the wave function into distinct components Oscillator potential (symmetric/asymmetric) and energy levels Interaction with light <y * m y > FTIR Absorption depends on dipole moment change Raman scattering depends on polarisability tensor Directional: strongest when parallel to electric vector of the incident light (Source: Wikipedia)

15 Delta Absorbance Molecular orientation in HPC films %rh 45 %rh 36 %rh Hydroxypropyl cellulose % 10% 20% 30% 40% 50% 60% Strain Parallel/ perpendicular in stretched film

Fracture in HPC film > 0.35 0.30-0.35 0.25-0.30 0.

16 Fracture in HPC film > < mm

17 Fracture in HPC film > < mm

18 Fracture in HPC film > < mm

19 Fracture in HPC film > < mm

20 Fracture in HPC film > < mm

21 Fracture in HPC film > < mm

22 Fracture in HPC film > < mm

- 970-933 - 924 Absorbance - 1126-1105 - 1153-1178 - 1050-1044 - 1002-1023 FTIR microscopy of maize starch Maize WT and

1 1200 1150 1100 1050 1000 950 900 Wavenumbers (cm -1 ) Iodine-stained micrograph and Cross-polarised micrograph FTIR

23 Absorbance FTIR microscopy of maize starch Maize WT and ae mutant wt 0.7 ae Wavenumbers (cm -1 ) Iodine-stained micrograph and Cross-polarised micrograph FTIR map of starch structure in maize sections monitored by the height of the 1002 cm -1 shoulder relative to the 1022 cm -1 peak maximum. Top: Wild type sections, Bottom: ae mutant maize 0.1 mm

24 Maize starch granules: Raman microscopy image - crystallinity wt ae 10 mm 10 mm

25 Maize starch granules: Raman microscopy image - branching wt ae 10 mm 10 mm

26 Maize starch granules: Raman microscopy image 860 cm -1 wt ae 10 mm 10 mm

27 Raman microscopy of WT maize starch granules protein map 153 carotene cm-1 combined map 153 starch ratio map 153 combined

Orientation in ae maize starch granules 20

28 Orientation in ae maize starch granules mm

29 Spectroscopy of polysaccharides Composition Orientation Interactions Location Composition of mutant Arabidopsis tissue Functional imaging of HPC film fracture IR and Raman investigation of starch granules in maize kernels

30 Thanks to IFR Mary Parker Yuri Alexeev Vic Morris Reg Wilson JIC Paul Derbyshire Alison Smith And you!

Supporting Information

Supporting Information Dauvillée et al. 10.1073/pnas.0907424106 Fig. S1. Iodine screening of the C. cohnii mutant bank. Each single colony was grown on rich-medium agar plates then vaporized with iodine.