The development of cells specialized for water conduction or support is a striking innovation of plants that has enabled them to colonize land. The NAC transcription factors regulate the differentiation of these cells in vascular plants. However, the path by which plants with these cells have evolved from their nonvascular ancestors is unclear. We investigated genes of the moss Physcomitrella patens that encode NAC proteins. Loss-of-function mutants formed abnormal water-conducting and supporting cells, as well as malformed sporophyte cells, and overexpression induced ectopic differentiation of water-conducting-like cells. Our results show conservation of transcriptional regulation and cellular function between moss and Arabidopsis thaliana water-conducting cells. The conserved genetic basis suggests roles for NAC proteins in the adaptation of plants to land.

The secondary cell walls of xylem cells, including vessel elements, provide mechanical strength and contribute to the conduction of water and minerals. VASCULAR-RELATED NAC-DOMAIN7 (VND7) is a NAC-domain transcription factor that regulates the expression of genes required for xylem vessel element formation. Transient expression assays using 68 transcription factors that are expressed during xylem vessel differentiation showed that 14 transcription factors, including VND1-VND7, are putative positive regulators of VND7 expression. Electrophoretic mobility shift assays revealed that all seven VND proteins bound to the VND7 promoter region at its SMBE/TERE motif, indicating that VND7 is a direct target of all of the VND transcription factors. Overexpression of VND1-VND5, GATA12 and ANAC075, newly identified transcription factors that function upstream of VND7, resulted in ectopic xylem vessel element formation. These data suggest that VND7 transcription is a regulatory target of multiple classes of transcription factors.

The Aurora kinase family is a well-characterized serine/threonine protein kinase family that regulates different processes of mitotic events. Although functions of animal and yeast Aurora kinases have been analyzed, plant aurora kinases were not identified and characterized. We identified three Aurora kinase orthologs in Arabidopsis thaliana and designated these as AtAUR1, AtAUR2, and AtAUR3. These AtAURs could phosphorylate serine 10 in histone H3, in vitro. Dynamic analyses of GFP-fused AtAUR proteins revealed that AtAUR1 and AtAUR2 localized at the nuclear membrane in interphase and located in mitotic spindles during cell division. AtAUR1 also localized in the cell plates. AtAUR3 showed dot-like distribution on condensed chromosomes at prophase and then localized at the metaphase plate. At late anaphase, AtAUR3 is evenly localized on chromosomes. The localization of AtAUR3 during mitosis is very similar to that of phosphorylated histone H3. Interestingly, an overexpression of AtAUR3 induces disassembly of spindle microtubules and alteration of orientation of cell division. Our results indicate that plant Aurora kinases have different characters from that of Aurora kinases of other eukaryotes.

Transgenic BY-2 cells stably expressing a GFP (green fluorescent protein)-tubulin fusion protein (BY-GT16) were subcultured in a modified Linsmaier and Skoog medium. The BY-GT16 cells could be synchronized by aphidicolin and the dynamics of their microtubules (MTs) were monitored by the confocal laser scanning microscopy (CLSM). We have succeeded in investigating the mode of reorganization of cortical MTs at the M/G1 interface. The cortical MTs were initially organized in the perinuclear regions and then they elongated to reach the cell cortex, forming the bright spots there. Subsequently, the first cortical MTs rapidly elongated from the spots and they were oriented parallel to the long axis towards the distal end of the cells. Around the time when the tips of the parallel MTs reached the distal end, the formation of transverse cortical MTs followed in the cortex near the division site, as we had previously suggested [Hasezawa and Nagata (1991) Bot. Acta 104: 206, Nagata et al. (1994) Planta 193: 567]. It was confirmed in independent observations that the appearance of the parallel MTs was followed by the appearance of the transverse MTs in each cell. We found that the transverse MTs spread through the whole cell cortex within about 20-30 min, while the parallel MTs disappeared. The significance of these observations on the mode of cortical MT organization is discussed.

It is a well-known hypothesis that cortical microtubules control the direction of cellulose microfibril deposition, and that the parallel cellulose microfibrils determine anisotropic cell expansion and plant cell morphogenesis. However, the molecular mechanism by which cortical microtubules regulate the orientation of cellulose microfibrils is still unclear. To investigate this mechanism, chemical genetic screening was performed. From this screening, 'SS compounds' were identified that induced a spherical swelling phenotype in tobacco BY-2 cells. The SS compounds could be categorized into three classes: those that disrupted the cortical microtubules; those that reduced cellulose microfibril content; and thirdly those that had neither of these effects. In the last class, a chemical designated 'cobtorin' was found to induce the spherical swelling phenotype at the lowest concentration, suggesting strong binding activity to the putative target. Examining cellulose microfibril regeneration using taxol-treated protoplasts revealed that the cobtorin compound perturbed the parallel alignment of pre-existing cortical microtubules and nascent cellulose microfibrils. Thus, cobtorin could be a novel inhibitor and an attractive tool for further investigation of the mechanism that enables cortical microtubules to guide the parallel deposition of cellulose microfibrils.