Abstract
Localisation of metabolites in sorghum coleoptiles using Raman hyperspectral imaging analysis was compared in wild type plants and mutants that lack cyanogenic glucosides. This novel method allows high spatial resolution in situ localization by detecting functional groups associated with cyanogenic glucosides using vibrational spectroscopy. Raman hyperspectral imaging revealed that dhurrin was found mainly surrounding epidermal, cortical and vascular tissue, with the greatest amount in cortical tissue. Numerous “hotspots” demonstrated dhurrin to be located within both cell walls and cytoplasm adpressed towards the plasmamembrane and not in the vacuole as previously reported. The high concentration of dhurrin in the outer cortical and epidermal cell layers is consistent with its role in defence against herbivory. This demonstrates the ability of Raman hyperspectral imaging to locate cyanogenic glucosides in intact tissues, avoiding possible perturbations and imprecision that may accompany methods that rely on bulk tissue extraction methods, such as protoplast isolation.
Highlights
Leaf composition in the past has typically been analysed by homogenizing whole leaves, or parts of leaves, removing the ability to distinguish between relative changes in composition of the different cell types present and rely on the assumption that the leaves are uniform in the distribution of chemicals across the leaf blade (e.g.1–3)
This band constitutes an unmistakable marker of cyanogenic glucosides, as the band occurs in an otherwise “quiet” region of the biological spectrum that is not populated by other bands of biological origins
Not unique to cyanogenic glucosides and found in the spectrum of other cell components, such as the phenolic lignin polymer of the cell wall, these bands from the C-C and C-H groups are very strong in the spectrum of cyanogenic glucosides and are likely to be observed together with the band from the nitrile moiety if dhurrin is present in the plant tissue
Summary
Leaf composition in the past has typically been analysed by homogenizing whole leaves, or parts of leaves, removing the ability to distinguish between relative changes in composition of the different cell types present and rely on the assumption that the leaves are uniform in the distribution of chemicals across the leaf blade (e.g.1–3). Cyanogenic glucosides are an important group of specialized metabolites that are hydrolysed with concomitant release of toxic hydrogen cyanide when mixed with specific β-glucosidases. Cyanogenesis is prevented by spatial separation at the organelle or tissue level of cyanogenic glucosides and the specific β-glucosidase enzymes required for their hydrolysis with the actual arrangement varying between species[18]. Disruption of this spatial separation e.g. by a chewing insect results in release of toxic hydrogen cyanide[18]. We combine Raman microspectroscopy with multivariate image analysis creating Raman hyperspectral imaging to visualize the concentration and distribution of cyanogenic glucosides in planta at sub-cellular spatial resolution, without the need for stains or chemical fixatives
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