Elucidation of molecular mechanism of habituation of cultured plant cells

在这项研究中，我们帮助阐明了烟草2B-13细胞生长素习服的分子机制。事实上，烟草BY-2细胞的增殖需要生长素，而2B-13细胞的生长不需要生长素。经过各种试验，我们发现添加2B-13细胞的培养滤液可以诱导生长素饥饿的烟草BY-2细胞的细胞分裂。然而，诱导细胞分裂的活性因子没有被归因于较低分子质量的分子，如植物激素，但活性部分被发现是较高分子质量的糖蛋白。通过羟基磷灰石层析、亲和层析和凝胶过滤等方法对该糖蛋白进行了纯化，结果表明该糖蛋白可能是一种相对分子质量为30 kDa的单一蛋白。另一方面，在烟草BY-2细胞培养滤液中寻找类似的因子发现，凝胶过滤后有两种糖蛋白。有趣的是，这些糖蛋白的分子量分别为25 kDa和40 kDa，与上面提到的30 kDa糖蛋白明显不同。由于到目前为止还没有发现可以被生长素取代的高相对分子质量的蛋白质，这些蛋白质应该是相当新的。确定这些蛋白质位于生长素信号通路的哪一步是很有趣的。另一方面，这三种蛋白质之间的关系也很有趣，这可以通过对这些蛋白质的氨基酸序列的测定来回答，这一点正在研究中。

In this study we have conduced the elucidation of the molecular mechanism of auxin-habituation using tobacco 2B-13 cells. In fact, tobacco BY-2 cells require auxin for their proliferation, while 2B-13 cells do not require it for growth. After various kinds of trials, we found that the addition of culture filtrates from the 2B-13 cells could induce cell division in the auxin-starved tobacco BY-2 cells. However, an active factor for inducing cell division was not ascribed to lower molecular mass molecules such as plant hormones, but the active fractions was found to be higher molecular mass glycoproteins. Thus, purification of this active fraction from the culture filtrates was conducted by hydroxyapatite chromatography, affinity chromatography and gel filtration, which revealed that the glycoprotein was ascribed possibly to a single protein of the molecular mass of 30 kDa in SDS-PAGE. On the other hand, seeking for similar factors in the culture filtrates of tobacco BY-2 cells revealed that there were two glycoproteins after the gel filtration. Intriguingly, these glycoproteins had molecular masses of 25 kDa and 40 kDa, respectively, which were apparently different from the 30 kDa glycoprotein mentioned above.Since such high molecular mass proteins that could be replaced with auxin have not been identified thus far, these proteins should be quite novel. It is intriguing to determine at which step of the auxin signaling pathway these proteins would be located. On the other hand, relationship among these three proteins is also interesting, which could be answered by the determination of amino acid sequences of these proteins, which is under study.