MYB3R-mediated and cell cycle-dependent transcriptional regulation of a tobacco ortholog of SCARECROW-LIKE28 in synchronized cultures of BY-2 cells

Although it is well known that hierarchical transcriptional networks are essential for various aspects of plant development and environmental response, little has been investigated about whether and how they also regulate the plant cell cycle. Recent studies on cell cycle regulation in Arabidopsis thaliana identified SCARECROW-LIKE28 (SCL28), a GRAS-type transcription factor, that constitutes a hierarchical transcriptional pathway comprised of MYB3R, SCL28 and SIAMESE-RELATED ( SMR). In this pathway, MYB3R family proteins regulate the G2/M-specific transcription of the SCL28 gene, of which products, in turn, positively regulate the transcription of SMR genes encoding a group of plantspecific inhibitor proteins of cyclin-dependent kinases. However, this pathway with a role in cell cycle inhibition is solely demonstrated in A. thaliana, thus leaving open the question of whether and to what extent this pathway is evolutionarily conserved in plants. In this study, we conducted differential display RT-PCR on synchronized Nicotiana tabacum (tobacco) BY-2 cells and identified several M-phase-specific cDNA clones, one of which turned out to be a tobacco ortholog of SCL28 and was designated NtSCL28. We showed that NtSCL28 is expressed specifically during G2/M and early G1 in the synchronized cultures of BY-2 cells. NtSCL28 contains MYB3R- binding promoter elements, so-called mitosis-specific activator elements, and is upregulated by a hyperactive form of NtmybA2, one of the MYB3R proteins from tobacco. Our study indicated that a part of the hierarchical pathway identified in A. thaliana is equally operating in tobacco cells, suggesting the conservation of this pathway across different families in evolution of angiosperm.