A Spatial-Temporal Analysis of Cellular Biopolymers on Leaf Blight-Infected Tea Plants Using Confocal Raman Microspectroscopy

Abstract

The objective of the present study was to characterize the temporal and spatial variation of biopolymers in cells infected by the tea leaf blight using confocal Raman microspectroscopy. We investigated the biopolymers on serial sections of the infection part, and four sections corresponding to different stages of infection were obtained for analysis. Raman spectra extracted from four selected regions (circumscribing the vascular bundle) were analyzed in detail to enable a semi-quantitative comparison of biopolymers on a micron-scale. As the infection progressed, lignin and other phenolic compounds decreased in the vascular bundle, while they increased in both the walls of the bundle sheath cells as well as their intracellular components. The amount of cellulose and other polysaccharides increased in all parts as the infection developed. The variations in the content of lignin and cellulose in different tissues of an individual plant may be part of the reason for the plant’s disease resistance. Through wavelet-based data mining, two-dimensional chemical images of lignin, cellulose and all biopolymers were quantified by integrating the characteristic spectral bands ranging from 1,589 to 1,607 cm–1, 1,087 to 1,100 cm–1, and 2,980 to 2,995 cm–1, respectively. The chemical images were consistent with the results of the semi-quantitative analysis, which indicated that the distribution of lignin in vascular bundle became irregular in sections with severe infection, and a substantial quantity of lignin was detected in the cell wall and inside the bundle sheath cell. In serious infected sections, cellulose was accumulated in vascular bundles and distributed within bundle sheath cells. In addition, the distribution of all biopolymers showed that there was a tylose substance produced within the vascular bundles to prevent the further development of pathogens. Therefore, confocal Raman microspectroscopy can be used as a powerful approach for investigating the temporal and spatial variation of biopolymers within cells. Through this method, we can gain knowledge about a plant’s defense mechanisms against fungal pathogens.