Molecular study on usage of unused available resources from northern trees

本文从分子水平上研究了北方树木皮层和木质部薄壁细胞对低温的适应机制，结果表明：北方树木皮层薄壁细胞通过细胞外冻结来适应低温。这些细胞通过获得对冷冻诱导的脱水的高耐受性，甚至可以适应液氮温度的冷冻。结果表明，第3组莱亚蛋白在冬季桑葚皮层薄壁细胞内质网中特异性积累。我们将该蛋白命名为WAP 27，并在植物和体外研究了其功能。转WAP 27基因拟南芥在低温锻炼后获得了比野生型更高的抗冻性。此外，纯化的WAP 27对低温敏感蛋白如LDH具有保护活性。另一方面，树木木质部薄壁细胞通过深度过冷来适应低于冰点的温度。北方树木的木质部薄壁细胞可以过冷到-60 关于我们 C.为了探讨水分在木质部薄壁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，发现了几个在植物中未曾报道过的冷诱导基因。我们通过北方印迹分析证实，这些基因的表达与过冷能力的增加平行。在使用山毛榉的蛋白质分析中，我们可以鉴定出几十个蛋白质点，这些蛋白质点与增加的过冷能力平行地特异性积累。在这些蛋白质中，我们纯化了几丁质酶样蛋白，这是已知的植物抗冻蛋白之一，抗冻活性检测结果为阴性。我们还分析了桦树木质部中的可溶性蛋白质。而皮层薄壁细胞通过细胞外冷冻来适应亚冰点温度，其积累量与皮层薄壁细胞相同。最后，我们从木质部（薄壁细胞）获得了粗提物，并检查了对水过冷的影响。结果表明，木质部粗提物在薄壁细胞中表现出较高的过冷度，从而提供了较高的过冷能力。现在，我们正在继续鉴定粗提物中可能提供高过冷的物质。我们的结果表明，来自表现出极高抗冻性的北方树木的冬季诱导基因和基因产物可能是开发高抗冻性作物的有用资源，也可以将其用于广泛的工业中的冷冻调节物质。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