构树韧皮部发育与主要代谢物累积的季节性规律

Phloem development, a vital natural science phenomenon, is connected with intracellular substance conversion and tree growth. Due to the rich secondary metabolites in phloem of Broussonetia papyrifera, they are a hinder in pulping and papermaking, on the other hand, these metabolites may also as potential resources to new materials and biological medicine. Hence, this project focuses on developing phloem tissue and puts forward new idea which combine with metabonomics and microtechniques to gain new insights into phloem formation in B. papyrifera. The metabolites are separated and identified by metabonomics method based on Gas chromatography and Mass spectrometry, then their seasonal variation is analyzed used unsupervised pattern recognition methods. The parenchymal cell is isolated by laser microdissection and its chemical features are analysed via GC-MS and High-performance liquid chromatography-Mass spectrometry (HPLC-MS) and the relationship between chemicals and phloem development is revealed. The structure of developing phloem is visualized in 3D by synchrotron X-ray microtomography. Polarized light, fluorescence light and electron microscope is employed to observed the cell structure and distribution, cell amount and its seasonal regular in phloem development. The kinds and content changes of Non-structural Carbohydrates（NSCs）is analyzed by HPLC and the changes in NSCs and main metabolites would be interlinked with the developmental changes in phloem. These results are meaning for illustration on carbon allocated in phloem development and foundation for resolve the mechanism of phloem cell formation in B. papyrifera.

韧皮部发育关系到细胞物质转化与树木生长，是非常重要的自然科学现象。构树韧皮部富含次生代谢产物，一方面阻碍制浆造纸，另一方面又是新材料和生物医药的潜在原料。本项目以构树发育中的韧皮部组织为对象，提出基于代谢组学与微技术相结合的研究思路，利用基于气质联用的代谢组学方法分离和鉴定代谢物，结合非监督模式识别方法确定代谢物的季节变化规律；采用激光显微切割技术精确分离薄壁细胞，依靠色谱质谱联用技术研究其化学组成，并探明它们与韧皮部发育之间的关系；基于同步辐射X射线显微CT技术构建韧皮部发育过程的可视化三维模型；用偏光、荧光显微镜、电镜等手段进行韧皮部发育中细胞结构和分布、细胞数量与季节规律的研究；借助高效液质联用技术研究非结构碳水化合物（NSCs）的种类与含量变化，揭示NSCs和主要代谢物与韧皮部发育之间的内在联系，对阐明韧皮部发育中碳素分配的规律具有重要意义，为构树韧皮部细胞构建机制的解析奠定基础。

项目针对构树韧皮部发育机制不清的科学问题，以1个生长季内韧皮部的形成过程为主线，结合代谢组学与显微技术的新思路，研究了构树韧皮部发育中在细胞和代谢物水平的季节变化规律，探索了韧皮部三维结构的可视化研究方法，分析了主要代谢物与韧皮部发育的相互关系。研究得出1）构树次生韧皮部由韧皮纤维，薄壁细胞，筛管分子，乳汁管和韧皮射线等细胞类型组成。在一个生长界限内细胞排列顺序为：乳汁管—韧皮薄壁细胞—筛管—韧皮纤维—筛管—韧皮薄壁细胞—乳汁管。韧皮部细胞具有周期性的季节变化。次生韧皮部细胞数量从5月至10月的生长季增加显著。韧皮部细胞的壁层加厚发生在生长季的后期（7-10月）。2）韧皮纤维量大，细胞壁厚，形似应拉木中的胶质木纤维；纤维长度最大值约7889μm，宽度最大值约19.5μm。韧皮纤维形态尺寸在生长季内是一个动态变化过程，纤维长度先减小后增大再减小，而其宽度先增加后减小。次生韧皮部化学组成主要由纤维素、半纤维素、淀粉等多糖组成，木质素含量极低。由于晶体的存在，使得韧皮部灰分含量增加。次生韧皮部中晶体主要出现在生长季后期，晶体的主要化学组成是一水合草酸钙和二水合草酸钙（少量，主要出现在生长季后期）。韧皮纤维结晶度随生长季而增大，在11月达到最大值，约31.5%。3）构树韧皮部代谢物以糖类物质为主，占代谢物总量的55%-84%。各类代谢物的相对含量呈现较明显的季节变化规律。非结构性碳水化合物主要有蔗糖、葡萄糖和果糖。莽草酸和阿魏酸都是合成木质素的主要中间体，证明了韧皮部细胞壁的快速木质化过程起始于7月份。4）乳汁管的数量从3月至9月呈上升趋势，然后至生长季末期略有下降。构树乳汁中主要由有酸类、糖类、脂类组成，还有少量的PI3-激酶抑制剂和生物碱，糖类主要是蔗糖。5）初步建立了基于X射线显微扫描和三维结构重建相结合的韧皮部可视化方法。项目的研究结果不仅为丰富林木韧皮部发育的基础理论提供了理论依据，还为构树韧皮的高效利用奠定了基础。