Hormone-induced differentiation of BY-2 cultured tobacco cells ; development of a model system for differentiation of root-cap cells.

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In conventional medium containing auxin (2,4-D, 0.2mg1^<-1>), BY-2 cultured tobacco cells proliferate actively, forming simple cell clusters composed of 8 to 16 small cylindrical cells connected in tandem, and the plastids within the cells remain undifferentiated throughout the culture period. By contrast, in a modified medium that contain cytokinin (BA, 1mg1^<-1>) instead of auxin, BY-2 cells become large without active cell proliferation, and the plastids within the cells differentiate into amyloplasts. In addition to these characteristics (inactive cell proliferation, cell enlargement, amyloplast development), we found that the behavior of BY-2 cells in the auxin-depleted medium resembled to that of root-cap cells in that (i) they easily exfoliate with each other, (ii) exhibited short lifetime, and (iii) showed higher secretion activity. Four proteins specifically secreted by root-cap cell-like BY-2 cells were detected, and one of them (named SPCT44) was identified as secreted peroxidase by N-terminal peptide sequencing of the purified protein. For other three proteins, we could not determine N-terminal peptide sequence because N-terminal was blocked. Their identification by determining internal peptide sequence is now on progress. To analyze changes in the gene expression during differentiation of normal BY-2 cells to root-cap like ones, we performed ddRT-PCR and microarray analyses (collaboration with RIKEN), using mRNAs purified from BY-2 cells cultured 12 h in either conventional medium or modified medium, and identified several genes whose expression is activated according to differentiation of root-cap like cells. The results are opened in the BY-2 EST database by RIKEN (http://mrg.psc.riken.go.jp/strc/). I'd like to further analyze the similarity between root-cap cells and the root-cap cell-like BY-2 cells in the modified medium, by using molecular markers obtained through this research project.