Molecular study on usage of unused available resources from northern trees

我们在分子水平上研究了北方树木的皮层和木质部薄壁细胞的冷冻适应机制。北方树木的皮层薄壁细胞通过细胞外冷冻来适应亚冰冻温度。这些细胞通过获得对冷冻诱导的脱水的高度耐受性，即使在液氮温度下也能适应冷冻。我们发现第3组LEA蛋白在越冬桑树皮层细胞的内质网中特异地积累。我们将该蛋白命名为WAP27，并在植物体内和体外检测了其功能。与野生型植株相比，导入WAP27的转基因拟南芥在冷驯化后获得了更高的耐寒性。此外，纯化的WAP27对树种木质部薄壁细胞等冷冻敏感蛋白具有低温保护活性，并能通过深过冷适应过冻温度。北方树木的木质部薄壁细胞可以过冷-60…为了研究水分在木质部薄壁组织细胞中保持过冷的机制，我们分析了基因表达、蛋白质和可溶性糖的积累。通过对落叶松木质部基因的综合分析，我们发现了一些在植物中从未报道过的冷诱导基因。Northern印迹分析证实，这些基因的表达与过冷能力的增强是平行的。在用山毛榉进行蛋白质分析时，我们可以识别出几十个蛋白质斑点，这些蛋白质斑点随着过冷能力的增强而专门积累。在这些蛋白中，我们纯化了几丁质酶样蛋白，这是一种已知的植物抗冻蛋白，并进行了抗冻活性检测，结果为阴性。我们还对桦木木质部的可溶性蛋白质进行了分析。而细胞外冷冻适应亚冰冻温度的大脑皮层细胞的积累量与之相同。最后，我们获得了木质部(薄壁细胞)的粗提物，并考察了其对水过冷的影响。我们可以发现，在薄壁细胞中表现出高过冷度的木质部粗提物具有较高的水过冷能力。现在，我们正在继续鉴定粗提液中可能提供高过冷度的物质。我们的结果表明，来自北方树木的冬季诱导基因和基因产品具有极高的抗寒性，可能是开发高抗冻性作物的有用资源，以及将其用于广泛行业的冷冻调节物质。较少

We studied freezing adaptation mechanisms of northern trees including the cortical and xylem parenchyma cells at molecular level.Cortical parenchyma cells of the northern trees adapt to subfreezing temperatures by extracellular freezing. These cells can adapt freezing even to liquid nitrogen temperature by obtaining high tolerance to freezing-induced dehydration. We identified that group 3 LEA proteins were specifically accumulated in ER of the cortical parenchyma cells of mulberry during winter. We named this protein as WAP27 and examined the function both in planta and in vitro. Transgenic Arabidopsis plants introduced WAP27 obtained higher freezing tolerance after cold acclimation as compared with wild type plants. Furthermore, purified WAP27 had cryoprotective activity to freezing sensitive proteins, such as LDH.Xylem parenchyma cells of trees, on the other hand, adapt to subfreezing temperatures by deep supercooling. The xylem parenchyma cells from northern trees can supercool -60 … More C. In order to examine mechanisms that water keep supercooling in xylem parenchyma cells, we analyzed gene expression, accumulation of proteins and soluble sugars. With comprehensive gene analysis about xylem of Larch, we could find several genes that were never reported as cold-induced genes in plants. We confirmed by northern blot analysis hat these genes were expressed in parallel with increased supercooling ability. On protein analysis using beech, we could identified a few tens protein-spots that were specifically accumulated in parallel with increased supercooling ability. Among these proteins, we purified chitinase-like proteins, which are known as one of antifreezing proteins in plants, and antifreeze activity was examined with negative result. We also analyzed soluble proteins in xylem of birch. However, the accumulation was same with the cortical parenchyma cells that adapt to subfreezing temperature by extracellular freezing. Finally, we obtained crude extracts from xylem (parenchyma cells) and the effect to supercooling of water was examined. We could find that the crude extracts from xylem that exhibited high supercooling in the parenchyma cells provided high ability of supercooling to water. Now, we are continuing identifications of substances within crude extracts that may provide high supercooling.Our results showed that winter-induced genes and gene products from northern trees that exhibit extraordinary high freezing resistance may be useful resources for developing crops with high freezing resistance as well as for the use of them for freezing regulate substances in wide range of industries. Less