*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.

R1R2R3-Myb proteins represent an evolutionarily conserved class of Myb family proteins important for cell cycle regulation and differentiation in eukaryotic cells. In plants, this class of Myb proteins are believed to regulate the transcription of G2/M phase-specific genes by binding to common cis-elements, called mitosis-specific activator (MSA) elements. In Arabidopsis (Arabidopsis thaliana), MYB3R1 and MYB3R4 act as transcriptional activators and positively regulate cytokinesis by activating the transcription of KNOLLE, which encodes a cytokinesis-specific syntaxin. Here, we show that the double mutation myb3r1 myb3r4 causes pleiotropic developmental defects, some of which are due to deficiency of KNOLLE whereas other are not, suggesting that multiple target genes are involved. Consistently, microarray analysis of the double mutant revealed altered expression of many genes, among which G2/M-specific genes showed significant overrepresentation of the MSA motif and a strong tendency to be down-regulated by the double mutation. Our results demonstrate, on a genome-wide level, the importance of the MYB3R-MSA pathway for regulating G2/M-specific transcription. In addition, MYB3R1 and MYB3R4 may have diverse roles during plant development by regulating G2/M-specific genes with various functions as well as genes possibly unrelated to the cell cycle.

Plant cells are surrounded by rigid cell walls, and hence, their division is associated with a plant-specific mode of cytokinesis in which the cell plate, a new cell wall, is generated and separates 2 daughter nuclei. The successful execution of cytokinesis requires the timely activation of multiple regulatory pathways, which include the AtNACK1/ HINKEL kinesin-induced MAPK cascade and MYB3R1/4-mediated transcriptional activation of G2/M-specific genes. However, it remains unclear whether and how these pathways are functionally interconnected to each other. By analyzing enhancer mutations of myb3r4, here we found a close genetic interaction between the 2 pathways; a mutation in ANP3, which encodes MAPKKK (acting downstream of AtNACK1/HINKEL), strongly enhanced the defective cytokinesis observed in the myb3r4 mutant. This interaction may not be due to the direct activation of MYB3R1/4 by the MAPK cascade; rather, possibly to the downstream targets of these 2 signaling pathways, acting in close proximity. Our results showed that MYB3R1/4 may positively affect cytokinesis via multiple pathways, one of which may act independently from the KNOLLE-dependent pathway defined previously, and affect the downstream events that may also be under the control of the AtNACK1/HINKEL-mediated MAPK cascade.Unlike animals, plants typically have a rigid cell wall outside of each cell, which provides mechanical strength that sustains their shape and growth. Because of the presence of the cell wall, plants have evolved a unique mode of cytokinesis, which accompanies the generation of a new cell wall, called the cell plate. During cell division, a microtubule-based structure, the phragmoplast, appears between 2 separating daughter nuclei and fulfills functions that are essential for the accumulation and fusion of Golgi-derived vesicles at the equatorial region.1,2 The molecular mechanisms underlying the formation of the cell plate are largely unknown; however, recent genetic studies have revealed components of signaling pathways that are essential for the execution of cytokinesis in Arabidopsis thaliana. 2,3