| Pyrus, classified as one of Pyrus bretshneider Rehd, was originally planted in Dangshan County, Anhui Province, and now has widely grown in the north, northwest, Old Yellow River and other regions in China. In recent years, due to factors such as variety deterioration and poor field management, pear pulp stone cell content has increased, and the texture of the pulp has become rougher with more pomace. These changes have adversely affected pear flavor and quality. In order to control the stone cell content and the stone cell size, it is significant to investigate its fruit cell development, especially including the stone cell development process. The stone cell,s formation and development is closely related to lignin biosynthesis, lignin transportation, and lignin deposition in stone cell secondary wall. Therefore, we figure that lignin metabolism and the stone cell secondary wall thickening play important roles in the stone cell development of Pyrus bretshneideri.In the study, through analyzing and measuring main enzymes activity for lignin biosynthesis, lignin content, and stone content in different periods of pear fruit development, I attempted to investigate the relations between lignin biosynthesis and stone cell growth. Through observing scopic and ultra-microscopic structures of the stone cell development, and analyzing lignin,s deposition in stone cell wall, I illustrated mechanism of the stone cell secondary wall formation, and then establish techniques to deeply explore the developmental mechanism of stone cells. The article is trying to lay the foundation for deep study about pear stone cell,s development mechanism by extracting and purifying lignin from pear fruit and then analyzing its lignin structural characteristics and types.The major findings are as follows:1. During the fruit development of Pyrus bretshneideri, stone cell content peaked at 51 days post flowering (17.82%); lignin content peaked at 47 and 63 days post flowering (6.47%, 7.00%); respectively POD activity peaked at 31, 43 and 55 days post flowering (184.5U/g.min, 221.4U/g.min and 242.9U/g.min); CAD activity peaked at day 35 after flowering (384.6U/g.min); C4H activity also had three peaks , which respectively occurred at 23, 39 and 63 days post flowering(204.9U/g.min, 143.2U/g.min, and 159.1U/g.min). these peaks appeared in some order: enzyme activity, then lignin content, and last stone cell content.2. During the stone cell development, lignin deposition in stone cells was started from the outer cell wall, and extended from the secondary wall to the whole cell lumen. Lignin deposition with a style of numerous discrete granules occurred at the corner of the primary cell wall, and then extended to the complex heterogeneous granular intercellular layer. With cellulose microfibrils deposition at the S1, S2 layer and its parallel arrangement along the circumference, lignin still deposited with granular unevenly along the direction of microfibrils deposition from its inside. With the alternative arrangement between them, cellulose microfibrils of the S2 layer and lignin granules filled up with the whole cell lumen.Lots of pits in it came into being, when stone cell wall was just thickening, It was apparently observed that finally pits formed branched holes. Many pits in stone cell walls gradually shifted from the open state to the close state. At the same time, the capacity of those pits exchanging water, materials between adjacent cells and surrounding environments little by little receded, and last lost.3. The optimal experimental methods of lignin extraction and purification from Pyrusbretshneideri are as follows: to distill by 1:10 ratio of material to dissolvent at the constant temperature of 60℃for 1h is the suitable condition of pretreatments on Pyrus bretshneideri;To extract by 1:10 ratio of material to dissolvent at the constant temperature of 20℃for 18h is the suitable condition of the distillation of lignin of Pyrus bretshneideri.4. Its UV spectrum showed a weak absorption peak at 274-276 nm, and an absorption maximum at approximately 288 nm. The absorption maximum at 288nm is character istic of benzene rings, and may be attributed to the guaiacyl groups in MWL. The absorption peak at 274-276 nm is characteristic of a syringyl group, and its weak intensity suggests that MWL contains more guaiacyl units than syringyl units. The wide shoulder peak at﹥300 nm on the spectrum suggests the presence of conjugated systems in the MWL.The fingerprint region on its FTIR spectrum (1800-800cm-1) showed most additional characteristic absorption peaks. The peak at 1227cm-1 is attributed to C-O stretching of the aromatic skeletons in the syringyl units. The peak at 1269cm-1 is characteristic of the stretching absorption of a guaiacyl group coupled to a carbonyl group. The intensity ratio of A1269 to A1227 was 1.25. Together, the FTIR results indicate that the structure of MWL is guaiacyl- syringyl-lignin, with guaiacyl units predominant. This is consistent with the results of the UV analyses.The ~1H-NMR spectrum also showed a weak peak at 6.6 ppm, in addition to a peak at 7.0 ppm. These peaks also indicated that its lignin structure is G-S-lignin, consistent with the above UV and FTIR analyses. In addition, based on the proton ratio of aromatic ester and aliphatic ester of acetic acid, the ratio between phenolic hydroxyl groups and alcoholic hydroxyl groups in MWL was calculated as 4.9/29.8 =0.16. Therefore, phenolic hydroxyl groups account for 16% of the total hydroxyl groups, and alcoholic hydroxyl groups account for the remaining 84%. This ratio is similar to the value of G-S-lignin in triploid Chinese white popular, but MWL in the pulp of Pyrus bretshneideri contains more hydrocarbon protons (15.8%) than triploid Chinese white popular.In conclusion, lignin UV, IR, 1H-NMR spectra analyses indicated that the lignin types of Pyrus bretschneideri are of guaiaeyl-syringyl (GS) type and there are more guaiaeyl units than syringl units in its lignin.
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