Epicuticular waxes, which are found on the outer surface of plant cuticles, are difficult to study in vivo. To monitor the growth, development, and structural alterations of epicuticular wax layers, coherent anti-Stokes Raman scattering (CARS) might be used. CARS, as a Raman-based technique, not only provides structural insight but also chemical information by imaging the spatial distribution of Raman-active vibrations. Here, we present a comparative study using CARS and scanning electron microscopy to characterize the structure of epicuticular waxes. The ability of CARS to provide detailed structural information on the biologically important wax layer was detailed on the examples of cherry laurel (Prunus laurocerasus), hoya (Hoya carnosa), and ceriman/Swiss cheese plant (Monstera sp. aff. deliciosa). We anticipate that the work presented will open a doorway for online monitoring of formation and alterations of epicuticular wax layers.

The invasive unicellular green macroalga Caulerpa taxifolia has spread dramatically in the Mediterranean Sea over the last decades. Its success is based on rapid plug formation after wounding, to prevent the loss of cell content. This quick and efficient process involves the rapid transformation of the secondary metabolite caulerpenyne to the reactive 1,4-dialdehyde oxytoxin 2, which acts as a protein crosslinker. The main metabolites of the wound plug were identified as proteins, caulerpenyne derivatives, and sulfated polysaccharides. Because of a methodological deficit, however, the detailed distribution of the compounds within the wound plug of C. taxifolia was unknown. This study demonstrates the suitability of FT-Raman spectroscopy for the noninvasive in vivo determination of caulerpenyne and its derivatives, as well as β-carotene, from signals with special spectral features within the wound plug and the adjacent intact alga tissue, with a resolution of 100 μm. FT-Raman spectra allowed four different zones with distinct chemical compositions around the region of wounds to be characterized. Gradients of the investigated metabolites within the wound plug and the alga could be determined. Moreover, various caulerpenyne derivatives could be identified spectroscopically, and this has led to a mechanistic proposal for the internal and the external wound plug formation.

Caulerpa spp. form a polymer wound plug that seals their giant cells after mechanical injury, in order to prevent fatal loss of cell material. Initial mass spectrometry and Raman spectroscopy investigations revealed the involvement of the secondary metabolite caulerpenyne in wound sealing polymer formation. In this work, we introduce a comparative Raman spectroscopic study of the wound plug formation in the invasive Caulerpa taxifolia (Valh) Agardh, 1817 and the non-invasive Caulerpa prolifera J.V. Lamour. In both species, the enzymatic transformation of the main secondary metabolite caulerpenyne plays a key role in wound plug formation. An accumulation of caulerpenyne is observed at the inner border of the wound plugs. Furthermore, caulerpenyne and products resulting from its enzymatic transformation and co-polymerization with proteins are found within the wound plug. However, there are significant differences in the chemistry of the wound plugs between the species. The Raman spectra reveal a zonation of the wound plug of C. taxifolia into four chemically distinguishable regions, while that of C. prolifera consists of only three regions with specific chemical composition. These results explain differences in the morphology of the wound plug in both species.

Um Wachstumsvorgänge in Pilzen oder Umwelteinflüsse auf Pflanzen zu untersuchen, eignen sich Verfahren der Ramanspektroskopie und ‐mikroskopie. Sie machen in lebenden Organismen die Morphochemie sichtbar, durch die sich molekulare Ursachen von Funktions‐ und Strukturänderungen erschließen.