Elucidation of molecular mechanism of habituation of cultured plant cells

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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.