Label-free visualization of fruit lignification: Raman molecular imaging of loquat lignified cells

Nan Zhu, Di Wu, Kunsong Chen

Flesh lignification, leading to increased fruit firmness, has been reported in several kinds of fruit. Understanding the mechanisms underlying fruit lignification is important to optimize the postharvest storage strategies and reduce the quality deterioration of postharvest fruit. Especially cellular level investigation of lignin deposition in fruits provides novel insight for deciphering the mechanisms underlying fruit lignification. The primary objective of this study was to establish a procedure of using Raman microspectroscopy technique to depict fruit lignification at the cell level. Lignified cells, a special kind of cells contained high lignin content, were found abundantly scattered in red-fleshed 'Luoyangqing' loquat. Whereas these special lignified cells were barely detected in 'Baisha' loquat flesh. Dominant Raman bands of lignified cells were found primarily attributed to lignin (1664, 1628, 1603, 1467, and 1272 cm-1), cellulose (1383, 1124 and 1098 cm-1) and pectin (852 and 1740 cm-1). The band intensity correlation analysis indicated the peak at 1335 cm-1 assigned to either lignin or cellulose in previous works was related to lignin for the lignified cells. Multi-peaks Gaussian fitting successfully resolved the overlapped fingerprint peaks of lignin in 1550-1700 cm-1 into three independent peaks, which were assigned to different functional groups of lignin. Furthermore, the spatially resolved Raman images of lignified cells were generated, indicating that lignin and cellulose saturated the whole lignified cells, pectin mainly located in the cell corner, and the parenchyma cells contained little lignin. In addition, both phloroglucinol-HCl staining and autofluorescence analysis confirmed the results of lignin distribution of Raman microscopic analysis. A procedure for the simultaneous visualization of the main components of the flesh cells without labeling by high-resolution Raman microspectroscopy has been established. With Raman microscopic imaging technique, we can add a microscopic level to cell compositions, essential for a detailed molecular understanding of loquat lignification. Such method can be further used to chemically monitor the textural changes during the ripening process or postharvest storage of other fruits and vegetables.