The tobacco mitogen-activated protein kinase kinase kinase NPK1 regulates lateral expansion of the cell plate at cytokinesis. Here, we show that the kinesin-like proteins NACK1 and NACK2 act as activators of NPK1. Biochemical analysis suggests that direct binding of NACK1 to NPK1 stimulates kinase activity. NACK1 is accumulated specifically in M phase and colocalized with NPK1 at the phragmoplast equator. Overexpression of a truncated NACK1 protein that lacks the motor domain disrupts NPK1 concentration at the phragmoplast equator and cell plate formation. Incomplete cytokinesis is also observed when expression of NACK1 and NACK2 is repressed by virus-induced gene silencing and in embryonic cells from Arabidopsis mutants in which a NACK1 ortholog is disrupted. Thus, we conclude that expansion of the cell plate requires NACK1/2 to regulate the activity and localization of NPK1.

*G2/M phase-specific gene transcription in tobacco cells is mediated by R1R2R3-Myb transcriptional activators, NtmybA1 and NtmybA2, which bind to mitosis-specific activator (MSA) elements. We show here that two structurally related genes, MYB3R1 and MYB3R4, which encode homologs of NtmybA1 and NtmybA2, play a partially redundant role in positively regulating cytokinesis in Arabidopsis thaliana. The myb3r1 myb3r4 double mutant often fails to complete cytokinesis, resulting in multinucleate cells with gapped walls and cell wall stubs in diverse tissues. These defects correlate with the selective reduction of transcript levels of several G2/M phase-specific genes, which include B2-type cyclin (CYCB2), CDC20.1 and KNOLLE (KN). These genes contain MSA-like motifs in their promoters and were activated by MYB3R4 in transient expression assays in tobacco cells. The KN gene encodes a cytokinesisspecific syntaxin that is essential for cell plate formation. The cytokinesis defects of myb3r1 myb3r4 double mutants were partially rescued by KN gene expression from heterologous promoters. In addition, a kn heterozygous mutation enhanced cytokinesis defects resulting from heterozygous or homozygous mutations in the MYB3R1 and MYB3R4 genes. Our results suggest that a pair of structurally related R1R2R3-Myb transcription factors may positively regulate cytokinesis mainly through transcriptional activation of the KN gene.

Mitogen-activated protein kinase (MAPK) cascades play important roles not only in the transduction of extracellular signals but in the progression of the cell cycle. However, evidence for their role in cytokinesis is limited. Here, we show that a tobacco MAPK kinase kinase (MAPKKK), designated NPK1, is required for cytokinesis. The activity of NPK1 increases in the late M phase of the tobacco cell cycle. During expansion of a new cross-wall (cell plate) toward the cell cortex, NPK1 is consistently localized to the equatorial zone of the phragmoplast, the cytokinetic apparatus where the cell plate is formed. Expression of a kinase-negative mutant of NPK1 results in the generation of multinucleate cells with incomplete cell plates. Phragmoplasts can be formed, but its expansion toward the cell cortex is also blocked. Thus, our results indicate that the NPK1 MAPKKK is essential for the formation of the cell plate, especially for its lateral growth. Cytokinesis is the last essential step in distribution of genetic information to daughter cells and cytoplasmic partition. Compared with the earlier steps of cell cycle such as DNA replication and separation of chromosomes, the modes of cytokinesis have diverged depending on organisms (Field et al. 1999). In animal cells, cytokinesis is performed by constriction, that is by an outside-in mode. The contractile ring of actin and myosin anchored on the cell cortex is critical for the constriction. In contrast, cytokinesis in plant cells occurs in the reverse way (for review, see Gunning 1982;Staehelin and Hepler 1996;Heese et al. 1998;Smith 1999). At late anaphase, a plant-specific and complex array of microtubules (MTs) and microfilaments called the phragmoplast is formed at the spindle midzone in the center of the cell. Antiparallel MTs interdigitate at their plus ends in the equatorial zone of the phragmoplast. Fusion of Golgiderived vesicles that are proposed to be transported along the MTs toward this zone generates a membranous network, which eventually maturates into a new cross wall called the cell plate. The growth of the cell plate is accompanied with lateral expansion of the phragmoplast, which allows vesicle fusions to occur continuously at the peripheral region of the cell plate, resulting in the inside-out mode of cytokinesis.These processes must involve a number of proteins for their execution and regulation. Many proteins have been found to be localized to the phragmoplast or the growing cell plate, including kinesin-like proteins (Liu et al.

Myb transcription factors, which contain three imperfect repeats in the Myb domain, are evolutionarily conserved members of the Myb superfamily. Vertebrate Myb proteins with three repeats, c-Myb, A-Myb, and BMyb, play important roles at the G 1 /S transition in the cell cycle. In plants, this type of Myb protein controls the G 2 /M phase by activating or repressing the transcription of cyclin B genes and a variety of other G 2 /M phasespecific genes. In tobacco, two genes for Myb activators, NtmybA1 and NtmybA2, are transcriptionally controlled and are expressed specifically at the G 2 /M phase. As we showed here, in addition to the control at the transcriptional level, activity of NtmybA2 is also controlled at the post-translational level. We found that the transactivation potential of NtmybA2 is repressed by a regulatory domain located at its carboxyl terminus and that specific classes of cyclins A and B enhanced NtmybA2 activity possibly by relieving this inhibitory effect. Mutations at the 20 potential sites of phosphorylation by cyclin-dependent kinase (CDK) in NtmybA2 blocked the enhancing effects of the cyclins on NtmybA2 activity. Recombinant NtmybA2 was phosphorylated in vitro by a CDK fraction prepared from tobacco BY2 cells. The kinase activity for NtmybA2 in the CDK fraction was cell cycle-regulated in BY2 cells, peaking at the G 2 /M phase when the level of transcripts of cyclin B is maximal. Taken together, our data suggest that NtmybA2 is phosphorylated by a specific cyclin/CDK complex(es) at G 2 /M and that this phosphorylation removes the inhibitory effect of its C-terminal region, thereby activating NtmybA2 specifically at G 2 /M.In the eukaryotic cell cycle, molecular events occur in a well-defined and reproducible sequence. The periodic activation at the transcriptional level of genes that regulate the cell cycle is fundamental to this process. In animals, transcription of several genes induced at the G 1 /S transition is mainly controlled by the E2F/DP heterodimeric transcription factors (for review, see Ref. 1). E2F-binding sequences are found in promoters from many genes that are transcribed at around the time of G 1 /S transition. Both E2F and DP factors have recently been identified in plants (for review, see Ref.2), and some reports indicate that plant E2F/DP factors play a role at the G 1 /S transition (3, 4). By contrast, different mechanisms for G 2 /M phase-specific transcription have been proposed in plants and animals. In animal cells, G 2 /M phase-specific transcription is mainly regulated by two repressor elements known as cell cycle-dependent elements (CDE) and cell cycle gene homology region (CHR) (for review, see Ref. 5). These elements are found in the promoters of various G 2 /M phase-specific genes, which include cdc25C (6), polo-like kinase (7), aurora A (8), and cyclin B2 (9). Mutation of the CDE and CHR elements allows elevated transcription during G 1 and consequent loss of cell cycle-regulated expression. In plants, G 2 /M phase-specific genes do not contain these rep...

Plant B-type cyclin genes are expressed specifically in late G2-and M-phases during the cell cycle. Their promoters contain a common cis-acting element, called the MSA (M-specific activator) element, that is necessary and sufficient for periodic promoter activation. This motif also is present in the tobacco kinesin-like protein gene NACK1 , which is expressed with timing similar to that of B-type cyclin genes. In this study, we show that G2/M-phase-specific activation of the NACK1 promoter also is regulated by the MSA element, suggesting that a defined set of G2/M-phase-specific genes are coregulated by an MSA-mediated mechanism. In a search for MSA binding factors by yeast one-hybrid screening, we identified three different Myb-like proteins that interact specifically with the MSA sequence. Unlike the majority of plant Myb-like proteins, these Myb proteins, NtmybA1, NtmybA2, and NtmybB, have three imperfect repeats in the DNA binding domain, as in animal c-Myb proteins. During the cell cycle, the level of NtmybB mRNA did not change significantly, whereas the levels of NtmybA1 and A2 mRNAs fluctuated and peaked at M-phase, when B-type cyclin genes were maximally induced. In transient expression assays, NtmybA1 and A2 activated the MSA-containing promoters, whereas NtmybB repressed them. Furthermore, expression of NtmybB repressed the transcriptional activation mediated by NtmybA2. Our data show that a group of plant Myb proteins that are structurally similar to animal c-Myb proteins have unexpected roles in G2/M-phase by modulating the expression of B-type cyclin genes and may regulate a suite of coexpressed genes.