酸沉降侵蚀下马尾松ATPase活性以及内源信号物质的研究

The forest ecosystem is an important component of the terrestrial ecosystem and plays an important role in global environmental maintenance. Tree growth situation is of great concern under the influence of acid deposition. Masson pine (Pinus massoniana Lamb.) is a widely distributed native pioneer tree, which grows on the acid forest soils in south China. This tree grows rapidly and is economically important. This tree also shows an inheritable tolerance on environmental stresses, including acid deposition. To better understand the acid-resistance mechanism of Masson pine under the errosion of acid deposition, the representative factors (biomass, plant type factor, root factor and stress index) must be used to comprehensively evaluate the acid- tolerant pedigree in our country. This innovative research, selects the acid-tolerant and acid-sensitive species as a comparative research materials based on the evaluation results, with the field experiments in Chongqing TieshanPing and Nanjing Zijin Mountain (control group). The experiment will be used to build an simulated model to mimic the climate system. We expand the model to investigate the effects of increasing acidity on tissue respiration, ATP contents of masson pine roots and leaves so that the model will be suitable for any other species. We use the cytochemical technique to examine the H+-ATPase and Ca2+-ATPase activities of plasma membrane in the two acid-treated species. Western blotting will be employed to establish the direct correlation between the activity of H+-ATPase and its protein expression. We will apply Scanning Polarographic Electrode Technique (SPET) to measure the O2 fluxes in acid-stressed roots of the two pines. From energy metabolism and signal transduction, the relationships will be revealed from acid rain between the ATPase enzyme activity and rooting endocrine regulation mechanisms. The following information will be also targeted: (a) the chemical signals from the root tip cells and the respiration rates of leaf and root; changes of root respiration rate and its internal O2 fluxes; (b) H+-ATPase activity of cell plasma membrane (tonoplast) within roots and leaves, namely the proton pump activity on cell membrane, and cell acid resistance as well as ion compartmentation capacity; (c) PM H+-ATPase activity of masson pine and its respiration ability; the Ca2+-ATPase activity of plasma membrane, vacuolar membrane, nuclear membrane of masson pine and its signal transduction capacity; (d) PMH+-ATPase activity and its protein expression, were be explored. Based on these relationships, the effects of ATPase activity regulation and endogenous signaling molecules on the acid-resistance mechanism of masson pine will be scientifically clarified..

森林是陆地生态系统中最重要的组成部分，酸沉降影响下的树木生长状况备受关注。为了深入了解酸沉降侵蚀下马尾松耐酸机制，选择具有代表性且能真实反映马尾松耐酸性的评价指标综合评价我国不同马尾松家系的耐酸性。选用强耐酸和弱耐酸马尾松作为对比试材，采用在重庆市铁山坪林场和南京紫金山林地（对照组）野外实验，以及酸雨人工气侯箱模拟实验的方法，测定马尾松根系与叶片呼吸速率、ATP含量；采用细胞化学方法研究马尾松细胞膜H+-ATPase和Ca2+-ATPase活性；应用免疫印迹法研究马尾松质膜H+ -ATPase蛋白的表达；利用扫描极谱电极技术测定马尾松根系氧的内流速率；再利用高效液相色谱法和气-质联用技术揭示马尾松内源信号物质的时序变化。从能量代谢和信号转导角度，探究ATPase活性调控、内源信号分子以及马尾松耐酸性能之间的内在逻辑关系，科学阐明ATPase活性调控以及内源信号分子对马尾松耐酸机制的影响。

本课题，结合我国酸雨污染现状，从林业生理学、细胞生物学、分子生物学层面研究酸雨对马尾松ATPase活性以及内源信号物质的作用，综合评价我国马尾松家系的耐酸性，进一步揭示木本植物耐受酸沉降侵蚀的内在机制，研究内容如下：.（1）以35、38、76、78、79、80、88、89、90、114、115、116、117、119、147和151号16个高产稳定型家系马尾松为试验材料，采用根箱技术、室外模拟酸雨喷淋法，基于马尾松的部分生理参数（生物量、质膜相对透性、叶绿素含量），综合分析各家系耐酸能力差异，其中，35家系耐酸能力最强，89、90、115和151家系次之，而79、114和116号家系则相对较弱。.（2）选用35家系强耐酸和116号家系弱耐酸马尾松为试材，在重庆市铁山坪林场和南京紫金山林地（对照组），以及酸雨人工气候箱开展实验，测定马尾松根系与叶片呼吸速率、ATP 含量；采用细胞化学方法研究马尾松细胞膜H+-ATPase 和Ca2+-ATPase 活性；应用免疫印迹法研究马尾松质膜H+-ATPase 蛋白的表达；利用扫描极谱电极技术测定马尾松根系氧的内流速率。从能量代谢和信号转导角度，探究ATPase 活性调控、内源信号分子以及马尾松耐酸性能之间的内在逻辑关系，科学阐明ATPase 活性调控以及内源信号分子对马尾松耐酸机制的影响。.（3）建立了快速溶剂萃取(ASE)固相萃取(SPE) 液相色谱电喷雾串联质谱法(ASE-SPE-LC-ESI-MS/MS)测定马尾松组织内超痕量有机酸和内源激素的方法。建立的方法灵敏、可靠，适宜植物组织或根系分泌物中超痕量有机酸和内源激素活性分子的测定，用于植物组织生物活性分子（有机酸、内源激素）相互作用以及迁移机理的探究，尽管植物样本基质特别复杂。.（4）酸雨会导致土壤酸化，影响植物的根际环境。用Tessier连续萃取和根箱栽培技术研究马尾松幼苗酸性根际土壤铝(Al)形态分布。在马尾松酸性根际土壤，观察到氧化还原电位降低，以及溶解有机碳增加。根际土壤pH值从5.93减至3.42，加速有机酸的分泌。交换铝形态和有机铝形态相应地依赖于土壤pH值和有机碳。这些数据有助于理解马尾松酸性根际土壤中Al形态的迁移力和生物有效性。