Label-free visualization of lignin deposition in loquats using complementary stimulated and spontaneous Raman microscopy

Nan Zhu,Di Wu,Yifan Yang,Kunsong Chen,Minbiao Ji

doi:10.1038/s41438-019-0153-3

Abstract

The lignification triggered by biotic or abiotic stresses hardens fruits and vegetables and eventually influences their consumer appeal. Extensive prior efforts have been made to unveil the underlying mechanism of flesh lignification, primarily focused on its physicochemical and molecular biological properties. Nevertheless, most of these studies used destroyed and homogenized bulk tissues as analytes; as a result, potentially valuable spatial information was lost. In this study, the deposition of lignin in loquat flesh during lignification was visualized from the tissue level to the single-cell level by combining the advantages of stimulated Raman scattering (SRS) and spontaneous Raman microscopy using label-free in situ molecular imaging. SRS has the advantages of being fast and providing large-area chemical imaging to reveal the spatial heterogeneity of lignin and cell wall polysaccharide distribution in loquat flesh. After 2 days of storage at 0 °C, increased lignins were observed by large-area SRS imaging. In addition, microscopic SRS images of the flesh cells indicated that the increased lignins were trapped in the cell corner (CC) and middle lamella (ML). Furthermore, the compositional and structural features of lignified cells (LCs), CC and ML of loquat flesh were investigated by spontaneous Raman microscopy, and the results showed that the LCs were a combination of lignin, cellulose, and hemicellulose, whereas CC and ML showed only deposited lignin and pectin without cross-linked cellulose and hemicellulose. This result further suggests that the lignins in the CC and ML regions of loquats were later synthesized alone during postharvest storage. This innovative combination of SRS and spontaneous Raman microscopy allows the label-free macroscale and fine chemical imaging of plant cell walls and will enhance our fundamental understanding of the structures and functions of the plant cell wall.

Highlights

In recent decades, fruits have become an indispensable part of the family diet, and the household consumption of fruit has increased significantly
In combination with microscopic chemical imaging, three types of cells were distinguished, i.e., the parenchymal cell (PC), lignified cell (LC), and vascular bundle (VB), which were the primary cell types found in loquats
PC, LC, and VB were all detected as stimulated Raman scattering (SRS) signals of cell wall polysaccharides at 2900 cm−1, which is generated by C–H stretching

Summary

Introduction

Fruits have become an indispensable part of the family diet, and the household consumption of fruit has increased significantly. Textural changes in fruits during postharvest that convey various sensory properties are attracting a great deal of concern and interest from consumers. The most typical textural characteristics of postharvest fruits are softening and lignification (hardening)[1,2]. Fruit lignification leading to increased fruit or fruit pericarp firmness is an unusual phenomenon that occurs in several fruits during postharvest[11,12]. Pericarp lignification is triggered by impact damage, which enhances the incorporation of phenolics into the lignin and increases the pericarp firmness[13]. Another notable case is the substantial lignification of loquat flesh

Methods

Results

Conclusion