Unlike mammalian and yeast cells, little is known about how plants regulate G 1 progression and entry into the S phase of the cell cycle. In mammalian cells, a key regulator of this process is the retinoblastoma tumor suppressor protein (RB). In contrast, G 1 control in Saccharomyces cerevisiae does not utilize an RB-like protein.We report here the cloning of cDNAs from two Zea mays genes, RRB1 and RRB2, that encode RB-related proteins. Further, RRB2 transcripts are alternatively spliced to yield two proteins with different C termini. At least one RRB gene is expressed in all the tissues examined, with the highest levels seen in the shoot apex. RRB1 is a 96-kDa nuclear protein that can physically interact with two mammalian DNA tumor virus oncoproteins, simian virus 40 large-T antigen and adenovirus E1A, and with a plant D-type cyclin. These associations are abolished by mutation of a conserved cysteine residue in RRB1 that is also essential for RB function. RRB1 binding potential is also sensitive to deletions in the conserved A and B domains, although differences exist in these effects compared to those of human RB. RRB1 can also bind to the AL1 protein from tomato golden mosaic virus (TGMV), a protein which is essential for TGMV DNA replication. These results suggest that G 1 regulation in plant cells is controlled by a mechanism which is much more similar to that found in mammalian cells than that in yeast.Progression through the G 1 phase of the eukaryotic cell cycle is tightly regulated, allowing cells to integrate internal and external cues before initiating DNA replication and committing to a round of cell division. This process is governed by both positive-and negative-acting regulatory factors. Although substantial progress has been made in understanding the mechanisms that govern these events in yeast and mammals (reviewed in reference 61), relatively little is known about G 1 regulation in plants. The existence of cyclin-dependent kinases (Cdks) and their associated cyclin subunits in plants (reviewed in reference 14) suggests that at least some of the basic mechanisms which regulate the cell cycle have been conserved throughout eukaryotic evolution. However, identification of additional regulatory components of the plant cell cycle is clearly essential for understanding plant growth and development.In the yeast Saccharomyces cerevisiae, progression through the G 1 phase is regulated by the Cdk Cdc28 (50), which in conjunction with G 1 cyclins activates the heterodimeric Swi4/ Swi6 transcription factor (40), resulting in the transcriptional activation of genes necessary for G 1 progression and S-phase entry (12, 61). In mammalian cells, G 1 progression also depends upon a Cdk-cyclin-activated transcriptional control pathway. However, a major regulatory protein in this pathway, the retinoblastoma protein (RB), has not been found in yeast. RB is the 110-kDa product of the retinoblastoma susceptibility tumor suppressor gene and plays key roles in regulating both cell cycle progression through the G 1 ph...

WD40 repeat proteins similar to yeast MSI1 are conserved in animals and plants, in which they participate in complexes involved in chromatin metabolism. Although MSI1-like proteins are well characterised biochemically,their function in the development of multicellular eukaryotes is not well understood. We constructed Arabidopsis plants in which the AtMSI1 protein level was altered. Strong ectopic expression of AtMSI1 produced no visible altered phenotype, but reduction of AtMSI1 dramatically affected development. The primary shoot apical meristem was unable to develop organs after the transition to flowering. Flowers that developed on floral shoots from axillary meristems experienced a progressive loss of floral morphology,including a reduction in size of the petals and stamens and the development of carpel-like sepals. Ovule development was disrupted in all flowers, resulting in complete female sterility. Molecular analysis of the mutant plants revealed that AtMSI1 is required to maintain the correct temporal and organ-specific expression of homeotic genes, including AGAMOUS and APETALA2. In contrast, FAS1 and FAS2, which together with AtMSI1 form the chromatin assembly complex CAF-1, are not required for repression of these genes. Therefore, AtMSI1 has specific functions in addition to CAF-1-mediated chromatin assembly. Efficient formation of heterochromatin, but not methylation of centromeric DNA repeats, depends on AtMSI1 presence demonstrating a key role of AtMSI1 in maintenance of chromatin structure.

Chromatin assembly factor CAF-1 facilitates the formation of nucleosomes on newly replicated DNA in vitro. However, the role of CAF-1 in development is poorly understood because mutants are not available in most multicellular model organisms. Biochemical evidence suggests that FASCIATA1, FASCIATA2 and MSI1 form CAF-1 in Arabidopsis thaliana. Because fasciata mutants are viable, CAF-1 is not essential for cell division in plants. Arabidopsis CAF-1 mutants have defects in shoot apical meristems; in addition, CAF-1 is required to establish seedling architecture, leaf size and trichome differentiation. CAF-1 is needed to restrict branching of trichomes on rosette leaves. Increased trichome branching in CAF-1 mutants is not strictly correlated with increased nuclear DNA content. In addition, fas2 glabra3 double mutants show an additive genetic interaction, demonstrating that CAF-1 acts genetically parallel to the GLABRA3-containing, endoreduplication-coupled trichome branching pathway. However, CAF-1 is often needed to restrict endoreduplication, because seedlings of most CAF-1 mutants have increased ploidy. Notably, in the Landsberg erecta background, loss of CAF-1 does not affect ploidy, demonstrating that loss of CAF-1 can be compensated in some Arabidopsis accessions. These results reveal that the functions of FAS1, FAS2 and MSI1 are not restricted to meristems, but are also needed to control genome replication at multiple steps of development.

In mammalian cells, the retinoblastoma (RB) protein regulates G , progression and functions through its association with various cellular proteins. Two closely related mammalian RB binding proteins, RbAp48 and RbAp46, share sequence homology with the Msil protein of yeast. MSll is a multicopy suppressor of a mutation in the lRA1jgene involved in the Ras-cAMP pathway that regulates cellular growth. Human RbAp48 is present in protein complexes involved in histone acetylation and chromatin assembly. We report the cloning of cDNAs encoding four plant RbAp48-and Msil -like proteins: one from tomato, LeMSIf, and three from Arabidopsis. Complementation studies confirm that LeMSl7 can function as a multicopy suppressor of the yeast ira1 mutant phenotype. The LeMSI1 protein localizes to the nucleus and binds to a 65-kD protein in wild-type as well as ripening inhibitor (rin) and Neverripe (Nr) tomato fruit. LeMSll also binds to the human RB protein and the RB-like RRBl protein from maize, indicating that this interaction is conserved between plants and animals.

In mammalian cells, the retinoblastoma (RB) protein regulates G1 progression and functions through its association with various cellular proteins. Two closely related mammalian RB binding proteins, RbAp48 and RbAp46, share sequence homology with the Msi1 protein of yeast. MSI1 is a multicopy suppressor of a mutation in the IRA1 gene involved in the Ras-cAMP pathway that regulates cellular growth. Human RbAp48 is present in protein complexes involved in histone acetylation and chromatin assembly. We report the cloning of cDNAs encoding four plant RbAp48- and Msi1-like proteins: one from tomato, LeMSI1, and three from Arabidopsis. Complementation studies confirm that LeMSI1 can function as a multicopy suppressor of the yeast ira1 mutant phenotype. The LeMSI1 protein localizes to the nucleus and binds to a 65-kD protein in wild-type as well as ripening inhibitor (rin) and Neverripe (Nr) tomato fruit. LeMSI1 also binds to the human RB protein and the RB-like RRB1 protein from maize, indicating that this interaction is conserved between plants and animals.