The maintenance of stem cells in defined locations is crucial for all multicellular organisms. Although intrinsic factors and signals for stem cell fate have been identified in several species, it has remained unclear how these connect to the ability to reenter the cell cycle that is one of the defining properties of stem cells. We show that local reduction of expression of the RETINOBLASTOMA-RELATED (RBR) gene in Arabidopsis roots increases the amount of stem cells without affecting cell cycle duration in mitotically active cells. Conversely, induced RBR overexpression dissipates stem cells prior to arresting other mitotic cells. Overexpression of D cyclins, KIP-related proteins, and E2F factors also affects root stem cell pool size, and genetic interactions suggest that these factors function in a canonical RBR pathway to regulate somatic stem cells. Expression analysis and genetic interactions position RBR-mediated regulation of the stem cell state downstream of the patterning gene SCARECROW.
Haploid spores of plants divide mitotically to form multicellular gametophytes. The female spore (megaspore) of most flowering plants develops by means of a well-defined programme into the mature megagametophyte consisting of the egg apparatus and a central cell. We investigated the role of the Arabidopsis retinoblastoma protein homologue and its function as a negative regulator of cell proliferation during megagametophyte development. Here we show that three mutant alleles of the gene for the Arabidopsis retinoblastoma-related protein, RBR1 (ref. 4), are gametophytic lethal. In heterozygous plants 50% of the ovules are aborted when the mutant allele is maternally inherited. The mature unfertilized mutant megagametophyte fails to arrest mitosis and undergoes excessive nuclear proliferation in the embryo sac. Supernumerary nuclei are present at the micropylar end of the megagametophyte, which develops into the egg apparatus and central cell. The central cell nucleus, which gives rise to the endosperm after fertilization, initiates autonomous endosperm development reminiscent of fertilization-independent seed (fis) mutants. Thus, RBR1 has a novel and previously unrecognized function in cell cycle control during gametogenesis and in the repression of autonomous endosperm development.
E2F transcription factors regulate genes expressed at the G1/S boundary of the cell division cycle in higher eukaryotes. Although animal E2F proteins and their target promoters have been studied extensively, little is known about how these factors regulate plant promoters. An earlier study identified two E2F consensus binding sites in the promoter of a Nicotiana benthamiana gene encoding proliferating cell nuclear antigen (PCNA) and showed that the proximal element (E2F2) is required for the full repression of PCNA expression in mature leaves. In this study, we examined the distal element (E2F1) and how it interacts with the E2F2 site to regulate the PCNA promoter. Gel shift assays using plant nuclear extracts or purified Arabidopsis E2F and DP proteins showed that different complexes bind to the two E2F sites. Mutation of the E2F1 site or both sites differentially altered PCNA promoter function in transgenic plants. As reported previously for the E2F2 mutation, the E2F1 and E2F1+2 mutations partially relieved the repression of the PCNA promoter in mature leaves. In young tissues, the E2F1 mutation resulted in a threefold reduction in PCNA promoter activity, whereas the E2F1+2 mutation had no detectable effect. The activity of E2F1+2 mutants was indistinguishable from that of E2F2 mutants. These results demonstrate that both E2F elements contribute to the repression of the PCNA promoter in mature leaves, whereas the E2F1 site counters the repression activity of the E2F2 element in young leaves.
In animal cells the progression of the cell cycle through G 1 /S transition and S phase is under the control of the pRB/E2F regulatory pathway. The E2F transcription factors are key activators of genes coding for several regulatory proteins and for enzymes involved in nucleotide and DNA synthesis. In this report we have detected the presence of E2F-like DNA binding activities in carrot nuclear extracts, and we have isolated a carrot cDNA (DcE2F) encoding a plant E2F homologue. The DcE2F gene is expressed in proliferating cells and is induced during the G 1 /S transition of the cell cycle. Supershift experiments using anti-DcE2F antiserum have confirmed that the DcE2F protein is a component of the carrot E2F-like nuclear activities. DNA binding assays have demonstrated that the DcE2F protein can recognize a canonical E2F cis-element in association with a mammalian DP protein. Furthermore, transactivation assays have revealed that DcE2F is a functional transcription factor that can transactivate, together with a DP partner, an E2F-responsive reporter gene in both plant and mammalian cells.Cell division in plants is mainly restricted to meristems and is regulated both temporally and spatially in response to plant growth regulators and environmental signals. Meristematic cell division occurs during the entire plant life, and because plant cells cannot migrate, control of cell proliferation is responsible for the formation of plant organs and structures (1). Remarkably, however, plant cells can readily undergo multiple cycles of endoreduplication, and additionally most of them are totipotent and can reprogram cell division even after completing their differentiation. These unique features suggest a unique flexibility in the control of the cell cycle in plants.In animal cells a pivotal role in the progression of cell cycle is played by the E2F family of transcription factors, which are key components of a critical checkpoint regulating entry of cells into S phase (2, 3). Progression through the G 1 and S phase of the cell cycle ultimately depends on the activation of genes coding for regulatory proteins and for the enzymes involved in nucleotide and DNA synthesis. The expression of many of these genes is largely under the control of the E2F family of transcription factors, which appear to be activated by multiple mitogenic signaling pathways. The E2F proteins bind to specific DNA sequences through a winged helix motif, forming prevalently heterodimers with distantly related partners of the DP (DRTF1 polypeptide) family (4). In mammalian cells six distinct E2F proteins associate with two different DP members and bind to similar DNA elements, which are conserved in the promoters of several genes that are activated in late G 1 and near the boundary G 1 /S. E2F gene targets include cell growth regulators such as cyclin A, Cdc2, c-Myc, and proliferating cell nuclear antigen and enzymes such as dihydrofolate reductase, thymidine kinase, ribonucleotide reductase, and DNA polymerase ␣ (5, 6). The importance of the E2F factors...
We describe methods for obtaining a quantitative description of RNA processing at high resolution in budding yeast. As a model gene expression system, we constructed tetON (for induction studies) and tetOFF (for repression, derepression, and RNA degradation studies) yeast strains with a series of reporter genes integrated in the genome under the control of a tetO7 promoter. Reverse transcription and quantitative real-time-PCR (RT-qPCR) methods were adapted to allow the determination of mRNA abundance as the average number of copies per cell in a population. Fluorescence in situ hybridization (FISH) measurements of transcript numbers in individual cells validated the RT-qPCR approach for the average copy-number determination despite the broad distribution of transcript levels within a population of cells. In addition, RT-qPCR was used to distinguish the products of the different steps in splicing of the reporter transcripts, and methods were developed to map and quantify 39-end cleavage and polyadenylation. This system permits pre-mRNA production, splicing, 39-end maturation and degradation to be quantitatively monitored with unprecedented kinetic detail, suitable for mathematical modeling. Using this approach, we demonstrate that reporter transcripts are spliced prior to their 39-end cleavage and polyadenylation, that is, cotranscriptionally.
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