Label-free Raman hyperspectral imaging analysis localizes the cyanogenic glucoside dhurrin to the cytoplasm in sorghum cells

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.

High-Fat Diet Impairs Mouse Median Eminence: A Study by Transmission and Scanning Electron Microscopy Coupled with Raman Spectroscopy

“…The optical feature at 2248 cm −1 represents a peculiar band located in the spectral region usually indicated as the silent zone due to the complete lack of Raman signals in biological specimen. This band is attributed to the cyanide group (-CN) [ 35 , 41 ], and it showed an evident higher peak in HFD spectra compared to CTRL ( Figure 5 E).…”

Dynamic metabolic solutions to the sessile life style of plants

“…The biosynthesis and storage of vanillin glucoside in phenyloplasts may represent a classical case of sub-cellular sequestration of phenolic compounds in specialized cellular compartments. Using Raman spectroscopy imaging of cross-sections of etiolated sorghum seedlings,123 the cyanogenic glucoside dhurrin was found to localize in the apoplast or cytoplasm, in the latter case most likely in phenyloplasts as previously suggested by Brillouet et al (2014) based on indirect histochemical data and electron microscopy images 117,124. Storage of dhurrin in phenyloplasts may have developed as a consequence of the chemical arms-race leading to high intracellular concentration of phytochemicals,125 which accumulate up to 30% of the dry weight in the tip of etiolated sorghum seedlings corresponding to molar concentrations 49,124,126…”

Physical energies to the rescue of damaged tissues

“…As in the universe, in biological organisms, rhythmic oscillations and syn-chronization of oscillatory patterns are an essential requisite for recognition and connectedness. Sophisticated approaches, including atomic force microscopy (AFM)[1-4], scanning tunneling microscopy (STM)[5,6], terahertz field microscopy (TFM)[7], and hyperspectral imaging (HSI)[8-10] are now providing a dynamic picture of the cellular environment at a nanoscale level, showing that mobile elements of the cyto- and nucleo-skeleton are dancing with patterns that display features of coherence, short- and long-range signal propagation, networking, and memory. Tubulin dimers, and microtubules are now emerging as the constituents of a highly dynamic web, acting both as a source for the generation and the context for the interplay of physical energies[5,6].…”