| Loquat belonging to the Rosaceae Eriobotrya is a peculiar perennial woody fruit tree native to southern China.The fruit of loquat is not resistant to storage and transportation,and it is prone to suffer lignification during postharvest storage,which significantly affects its commercial value and restricts its industrial development.At present,the related researches on postharvest lignification of loquat fruit are mainly focused on the analysis of physicochemical characteristics of the flesh tissues and investigating the expression patterns and transcriptional regulation of lignin biosynthesis related genes of loquat fruit during postharvest lignification.However,the specific sites of lignin accumulation,and the dynamic process of lignin accumulation,and the cell types in which lignification occurs during postharvest storage remain unclear.The cellular level mechanisms of postharvest lignification need to be clarified.The purpose of this study was to investigate the dynamic changes of spatial distribution of cell wall components from single cell level to multi-cell level in loquat flesh during postharvest storage and to identify the specific deposition sites of the newly synthesized lignin in flesh cells and construct the cellular lignin accumulation model for the loquat during lignification based on multi-component simultaneous molecular imaging and click-chemistry-based imaging techniques,so as to provide a research foundation and theoretical support for a visual understanding of the relationship between lignin accumulation and fruit firmness increment and rough taste,revealing the cell biological mechanism of postharvest lignification,and optimizing postharvest storage and preservation strategy.The main research contents and results are as follows:1.Multicomponent synchronous imaging and spatial distribution analysis of the lignified cells in loquat flesh.First,a special kind of cell with significantly higher lignin content than the surrounding parenchyma cells,named lignified cell,was found in the'Luoyangqing'loquat flesh.Confocal Raman microspectroscopy?CRM?imaging system combined with spectrogram analysis algorithm was used for the spectral response analysis,feature extraction,multiscale spectral fusion and spectrogram fingerprint mining.Multicomponent synchronous imaging of the lignified cell was performed which made the synchronous analysis the structure and spatial distribution of multi chemical components of lignified cell possible.Dominant Raman bands of lignified cells were found primarily attributed to lignin(1664,1628,1603,1467,1335 and 1272 cm-1)and cellulose(1383,1124 and 1098 cm-1).The two peaks at 1272 and 1335 cm-1 found in the Raman spectra of the lignified cell indicate that the lignin in the lignified cell of loquat fruit is in G/S type.Based on the micro-Raman hyperspectral images of lignified cell,it was found that the lignin was distributed throughout the lignified cells and the peripheral edge of the lignified cell contained stronger lignin Raman signal.The cellulose was evenly distributed in the lignified cells and pectin was distributed in the corner?CC?between the lignified cell and the surrounding parenchyma cels.2.Analysis of the spatial distribution and structural changes of cell wall components from macroscopic scale to single cell level in the loquat flesh.The complementary stimulated Raman scattering?SRS?and CRM imaging techniques were combined to capture the dynamic changes of cell wall polysaccharide and lignin distribution from macroscale to single cell level to investigate the compositional and structural variation in loquat cell.The SRS imaging revealed the heterogeneity in the spatial distribution of lignin and cell wall polysaccharides in loquat flesh.The lignified cell and vascular bundle presented strong lignin and cell wall polysaccharides SRS signals;however,no lignin SRS signal was detected in parenchyma cell,and the SRS signal of the cell wall polysaccharides was also weaker than that of lignified cells and vascular bundle cells.After 2 days of storage at 0°C,increased lignins were observed by large-area SRS imaging.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 cell,CC and ML of loquat flesh were investigated by CRM.The results indicate that the CC and ML only deposited lignin and pectin without cross-linked cellulose compared with the lignified cell.Moreover,the lignins in the CC and ML were found containing a higher content of aldehyde structures compared to lignified cells.According to the above results,it suggests that the lignins in the CC and ML regions of loquats were later synthesized alone during postharvest storage.3.The specific spatio-te mporal labeling of the ne wly synthesized lignin in loquat flesh during postharvest and the proposal of a cellular model for the specific accumulation of lignin during lignification.A mimic of coniferyl alcohol?CA?,CA-Alk,was synthesized,and its biological activity was verified.The system of specific labeling and imaging of the newly synthesized lignin in the cells of loquat flesh was constructed using click-chemistry-based imaging technique.The results show that the CA-Alk can be successfully incorporated into the newly synthesized lignin in loquat flesh,which makes it available for the spatial-temporal specific labeling and tracking of the newly synthesized lignin in the flesh cells during the feeding period.It was found that the lignified cells and vascular bundles in the fruitlet showed high activity in incorporating CA-Alk into the newly synthesized lignin during the precursor feeding period.Besides,both the secondary wall and the peripheral region of the lignified cell accumulate lignin.For the postharvest loquat fruit,the newly synthesized lignin were found specifically deposited into the CC and ML of the parenchyma cells near the vascular bundle;there's no newly synthesized lignin accumulation found in the lignified cells during the feeding period.The lignin staining of the flesh and the flesh microsections of postharvest loquat also showed that the CC and ML of the parenchyma cells near the vascular bundle are rich in lignin deposits. |
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