Molecular study on usage of unused available resources from northern trees

从分子水平研究了北方乔木皮层和木质部薄壁细胞的冰冻适应机制。北方乔木的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引起的脱水的高耐受性，甚至可以适应液氮温度的冷冻。我们发现，在桑树皮层薄壁细胞的内质网中，3组LEA蛋白在冬季特异性积累。我们将该蛋白命名为WAP27，并对其在植物体内和体外的功能进行了研究。引入WAP27的转基因拟南芥在冷驯化后的抗冻性高于野生型。此外，纯化后的WAP27对LDH等冷冻敏感蛋白具有冷冻保护活性。另一方面，树木的木质部薄壁细胞通过深度过冷来适应冰点以下的温度。为了探究水分保持木质部薄壁细胞过冷的机制，我们分析了水分保持木质部薄壁细胞过冷的基因表达、蛋白质积累和可溶性糖。通过对落叶松木质部的综合基因分析，我们发现了一些从未报道过的植物冷诱导基因。我们通过northern blot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点，这些蛋白质点是与过冷能力增加并行积累的。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。我们还分析了桦木木质部的可溶性蛋白。细胞外冷冻适应低温的皮层薄壁细胞，其积累量与适应低温的皮层薄壁细胞相同。最后，我们从木质部（薄壁细胞）中提取粗提物，并考察了水对过冷的影响。木质部粗提物在薄壁细胞中表现出较高的过冷性，对水分具有较高的过冷性。现在，我们正在继续鉴定粗提取物中可能提供高过冷性的物质。我们的研究结果表明，来自北方树木的具有非凡抗冻性的冬季诱导基因和基因产物可能是开发具有高抗冻性作物的有用资源，以及在广泛的工业中使用它们作为冷冻调节物质。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