枇杷果实采后木质化细胞形成机制的非标记原位分子成像研究

Chilling injury of lignification is commonly happened when loquat fruit is in postharvest cryopreservation, which affects the fruit’s commercial value seriously. Previous researches show that there are lignified cells developed during the lignification of loquat fruit. However, the variation mechanism of cell molecules at the single cell level during the development of lignified cells is not clear. The visualization of the developing mechanism is required. Common microscopy cannot obtain the information of cell molecules, while the research of molecular structure is based on the extraction and purification processes, which can destroy the cells’ microstructure. Both of them cannot study the cell molecules in situ and micro-visualize cell molecules quantitatively. The present project will use Raman and IR microscopic hyperspectral imaging, which have the advantages of containing molecular vibration information in Raman and IR spectra and providing their micro-imaging in a label-free and in-situ way. Mathematical analysis algorithms for spectral resolution, dimension reduction, and data fusion will be developed and used to analyze the information of molecular structures contained in the spectral data of lignified cells in-depth. The molecular changes of lignified cells in situ at different development stages will be analyzed. The relationships of changes between different molecules will be explored during the development of lignified cells. The development mechanism of lignified cells will be clarified at the cellular level from the perspective of the molecular structure. The molecular changes during the development of lignified cells will be visualized in micro-images in a label-free way, so that the development mechanism of lignified cells in postharvest loquat fruit can be understood visually. A new basis will be provided for optimizing the storage and preservation technology of postharvest loquat fruit and improving its quality.

枇杷果实采后低温贮藏易出现冷害木质化，严重影响果实的商品价值。前期研究表明，枇杷木质化伴随出现木质化细胞，但其形成过程中细胞分子在单细胞水平的变化机制还不明确，缺少相关机制的直观可视化表达。常规显微技术无法获得细胞分子信息，而基于提取纯化的分子结构研究则会破坏细胞结构，均无法实现细胞分子的原位研究和微观定量可视化表达。本项目拟应用拉曼及红外显微光谱成像技术，基于其光谱包含分子振动信息且可以非标记原位显微成像的优势，研究并利用解析、降维、融合等多种数据挖掘算法，深入分析和提取光谱信号包含的木质化细胞分子信息，原位研究木质化细胞形成过程的分子变化，探究木质化细胞形成中各分子间的相互变化关系，从分子结构角度在单细胞水平阐明木质化细胞形成机制，并对形成过程中细胞分子变化进行非标记微观可视化表达，以便更直观地理解枇杷果实采后木质化细胞的形成机制，并为优化采后贮藏保鲜工艺、提高果实品质提供新依据。

枇杷果实采后低温贮藏易出现冷害木质化，严重影响果实的商品价值。前期研究表明，枇杷木质化伴随出现木质化细胞，但其形成过程中细胞分子在单细胞水平的变化机制还不明确，缺少相关机制的直观可视化表达。本项目开展了枇杷果实采后木质化细胞形成机制的非标记原位分子成像研究。主要研究结果如下：（1）枇杷果实在贮藏期间的木质化过程中，果肉硬度、果肉木质素含量、果肉木质素染色面积占比、木质化细胞数量均呈上升趋势，且彼此间呈现显著正相关。（2）枇杷果肉木质素的主要积累在木质化细胞、维管束以及部分薄壁细胞的细胞壁中间层和角隅。果肉木质素染色区域面积的主要贡献为木质化细胞；部分薄壁细胞的细胞壁中间层和角隅的染色面积很小。（3）发现枇杷果实中的木质化细胞主要可以分为四种类型，提出木质化细胞的发育过程共分为四个阶段，并发现单个木质化细胞的发育过程和枇杷果实的贮藏阶段是两个独立的过程。（4）分析了枇杷果实木质化细胞在不同形成阶段的细胞壁成分的拉曼特征光谱，实现了木质素、纤维素、果胶和木质素特征官能团在木质化细胞不同形成阶段的可视化表达，并对木质化细胞的木质素拉曼成像结果进行了验证。（5）总结了细胞壁物质在木质化细胞形成过程中的积累规律，指出木质素和纤维素积累是木质化细胞形成的主要因素。（6）发现部分木质化细胞在实心化后，其周边的一些薄壁细胞也会开始积累木质素，且在其与实心木质化细胞接触的细胞壁处，先出现木质素和纤维素的拉曼信号。（7）实现了枇杷果肉细胞壁多糖和木质素在宏观尺度的细胞群成像，发现木质化细胞和维管束含有较高含量的细胞壁多糖和木质素，而薄壁细胞的细胞壁几乎检测不到木质素信号。（8）发现不同细胞区域的细胞壁物质谱峰存在差异。本研究结果有利于更直观地理解枇杷果实采后木质化细胞的形成机制，可以为深入探讨枇杷果实采后木质化机制提供理论支撑，并为优化枇杷果实采后贮藏保鲜工艺、提高果实品质提供新依据。

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