In multicellular organisms, temporal and spatial regulation of cell proliferation is central for generating organs with defined sizes and morphologies. For establishing and maintaining the postmitotic quiescent state during cell differentiation, it is important to repress genes with mitotic functions. We found that three of the Arabidopsis MYB3R transcription factors synergistically maintain G2/M-specific genes repressed in post-mitotic cells and restrict the time window of mitotic gene expression in proliferating cells. The combined mutants of the three repressor-type MYB3R genes displayed long roots, enlarged leaves, embryos, and seeds. Genome-wide chromatin immunoprecipitation revealed that MYB3R3 binds to the promoters of G2/M-specific genes and to E2F target genes. MYB3R3 associates with the repressor-type E2F, E2FC, and the RETINOBLASTOMA RELATED proteins. In contrast, the activator MYB3R4 was in complex with E2FB in proliferating cells. With mass spectrometry and pairwise interaction assays, we identified some of the other conserved components of the multiprotein complexes, known as DREAM/dREAM in human and flies. In plants, these repressor complexes are important for periodic expression during cell cycle and to establish a post-mitotic quiescent state determining organ size.

*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.

We have isolated 5 cDNA clones (din2, din6, din9, din10 and din11) corresponding to genes, the transcripts of which accumulated in leaves of Arabidopsis thaliana kept in the dark. These cDNA clones encode proteins similar to beta-glucosidase (EC 3.2.1.21, din2), asparagine synthetase (EC 6.3.5.4, din6), phosphomannose isomerase (EC 5.3.1.8, din9), seed imbibition protein (din10) and 2-oxoacid-dependent dioxygenases (din11). Accumulation of the transcripts from din6 and din10 occurred within 3 h after plants were transferred to darkness. The transcripts from din2, din9 and din11 were only detected after 24 h of dark treatment. We also observed the accumulation of the din transcripts in senescing leaves. Application of a photosynthesis inhibitor, 3-(3,4-dichlorophenyl)-1-1-dimethyl-urea, induced the expression of the din genes under illumination. Application of sucrose to detached leaves suppressed the accumulation of the din transcripts in the dark. These results indicate that expression of these genes partly depends on cellular sugar level. The sugar-modulated expression of the din genes suggests that dark-induced expression of these genes might be related to sugar starvation occurring in leaf cells in the dark, when the photosynthesis is hindered.

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.

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.

Increased cellular ploidy is widespread during developmental processes of multicellular organisms, especially in plants. Elevated ploidy levels are typically achieved either by endoreplication or endomitosis, which are often regarded as modified cell cycles that lack an M phase either entirely or partially. We identified GIGAS CELL1 (GIG1)/OMISSION OF SECOND DIVISION1 (OSD1) and established that mutation of this gene triggered ectopic endomitosis. On the other hand, it has been reported that a paralog of GIG1/OSD1, UV-INSENSITIVE4 (UVI4), negatively regulates endoreplication onset in Arabidopsis thaliana. We showed that GIG1/OSD1 and UVI4 encode novel plant-specific inhibitors of the anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase. These proteins physically interact with APC/C activators, CDC20/FZY and CDH1/FZR, in yeast two-hybrid assays. Overexpression of CDC20.1 and CCS52B/FZR3 differentially promoted ectopic endomitosis in gig1/osd1 and premature occurrence of endoreplication in uvi4. Our data suggest that GIG1/OSD1 and UVI4 may prevent an unscheduled increase in cellular ploidy by preferentially inhibiting APC/CCDC20 and APC/CFZR, respectively. Generation of cells with a mixed identity in gig1/osd1 further suggested that the APC/C may have an unexpected role for cell fate determination in addition to its role for proper mitotic progression.

The 6.5-kilobase mre region at 71 min in the Escherichia coli chromosome map, where genes involved in formation of a rod-shaped cell form a gene cluster, was analyzed by in vivo protein synthesis in a maxicell system and by base sequencing of DNA. An open reading frame that may code for a protein with an Mr of about 37,000 on sodium dodecyl sulfate-polyacrylamide gels was found and was correlated with the mreB gene. N-terminal amino acid sequencing of the hybrid mreB-lacZ protein confirmed the production by mreB of a protein of 347 amino acid residues with a molecular weight of 36,958. The amino acid sequence of this protein deduced from the DNA sequence showed close similarity with that of a protein of theftsA gene which is involved in cell division of E. coli. Three other contiguous genes that formed three proteins with Mrs of about 40,000, 22,000, and 51,000, respectively, were detected downstream of the mreB gene by in vivo protein synthesis. The mreB protein and some of these three proteins may function together in determination of cell shape.Two regions called mrd (15) and mre (11) on the Escherichia coli chromosome involve clusters of genes responsible for determination of the cell shape and the sensitivity of cell growth to an amidinopenicillin, mecillinam. The mrd region located at 15 min on the E. coli chromosome map involves two genes for formation of the rod shape of the cell, mrdA (= pbpA), which codes for penicillin-binding protein 2, a peptidoglycan synthetase, and mrdB (= rodA), which codes for the RodA protein which may also be necessary for functioning of the mrdA protein (5). The mre region located at 71 min on the chromosome map involves genes that probably function together in shape determination and mecillinam sensitivity of the cell (17). Previously, we reported preliminary results of gene analysis of the apparent 7-kilobase (kb) mre region neighboring the fabE gene (17). The gene we called mreB (11), which may be allelic to envB (18) and in which the mutation mre-129 causing a round cell and supersensitivity to mecillinam is located, was cloned in a 2.8-kb DNA fragment. However, we found that another mutation, mre-678, causing a round cell, resistance to mecillinam, and overproduction of penicillin-binding proteins lBs and 3 was due to deletion of a 5.2-kb DNA fragment extending from the mreB gene and could not be complemented by the 2.8-kb fragment (17).The present report describes the in vivo identification of the protein products of the genes located in the mre region (the size of the SaII fragment is corrected to 6.5 kb from that in the previous report) by using a maxicell system (13) MATERIALS AND METHODSBacterial strains. E. coli K-12 strain CSR603 (uvrA6 recAl phr-J thr leu pro his thi arg lac gal ara xyl mtl rpsL) used for protein synthesis in the maxicell system was obtained from B. J. Bachmann, Yale University School of Medicine. Strain JM109 (Alac-proAB recAl endAl gyrA96 thi hsdRJ7 supE44 relAl A-F' traD36 proAB lacIq AlacZMJ5) used for DNA sequencing experiments was obtained...

SummaryWe have isolated a delayed-leaf-senescence mutant, designated dls1, from an Arabidopsis T-DNA line. Leaf senescence progresses more slowly in the dls1 mutant than in the wild-type plant in both age-dependent and dark-induced senescence. Genetic analysis revealed that the dls1 is a monogenic recessive mutation that cosegregated with the T-DNA insertion. Isolation of DNA flanking the T-DNA revealed that the T-DNA was inserted into the fourth intron of the AtATE1 gene, which encodes arginyl-tRNA:protein arginyltransferase (EC. 2.3.2.8, R-transferase), a component of the N-end rule proteolytic pathway in yeast and mammals that transfers arginine to the N-terminus of proteins with N-terminal glutamyl or aspartyl residues. AtATE1 transcripts were not detectable in the dls1 mutant by RT-PCR analysis. Introduction of a wild-type AtATE1 gene into the dls1 mutant complemented the dls1 phenotype. We also showed using a transient expression assay system, that the dls1 mutation results in a decreased degradation of proteins with Asp or Glu at their N-termini, and that the introduction of the wild-type AtATE1 gene reverses this deficiency. These results suggest that the normal progression of leaf senescence requires R-transferase activity, and that proteolysis by the N-end rule pathway has an important physiological function in the progress of leaf senescence in plants.