SIAMESE, a gene controlling the endoreduplication cell cycle in Arabidopsis thaliana trichomes

Cell differentiation is generally tightly coordinated with the cell cycle, typically resulting in a nondividing cell with a unique differentiated morphology. The unicellular trichomes of Arabidopsis are a well-established model for the study of plant cell differentiation. Here, we describe a new genetic locus, SIAMESE (SIM), required for coordinating cell division and cell differentiation during the development of Arabidopsis trichomes (epidermal hairs). A recessive mutation in the sim locus on chromosome 5 results in clusters of adjacent trichomes that appeared to be morphologically identical 'twins'. Upon closer inspection, the sim mutant was found to produce multicellular trichomes in contrast to the unicellular trichomes produced by wild-type (WT) plants. Mutant trichomes consisting of up to 15 cells have been observed. Scanning electron microscopy of developing sim trichomes suggests that the cell divisions occur very early in the development of mutant trichomes. WT trichome nuclei continue to replicate their DNA after mitosis and cytokinesis have ceased, and as a consequence have a DNA content much greater than 2C. This phenomenon is known as endoreduplication. Individual nuclei of sim trichomes have a reduced level of endoreduplication relative to WT trichome nuclei. Endoreduplication is also reduced in dark-grown sim hypocotyls relative to WT, but not in light-grown hypocotyls. Double mutants of sim with either of two other mutants affecting endoreduplication, triptychon (try) and glabra3 (gl3) are consistent with a function for SIM in endoreduplication. SIM may function as a repressor of mitosis in the endoreduplication cell cycle. Additionally, the relatively normal morphology of multicellular sim trichomes indicates that trichome morphogenesis can occur relatively normally even when the trichome precursor cell continues to divide. The sim mutant phenotype also has implications for the evolution of multicellular trichomes.     

Trichome morphogenesis: a cell-cycle perspective

Arabidopsis leaf hairs (trichomes) are polyploid epidermal cells with a predictable branching pattern. More than 15 genes have been identified that are involved in the regulation of branching. The cloning of the ZWICHEL, ANGUSTIFOLIA and STICHEL genes points to two mechanistic aspects of branch formation: (i) a role of the microtubule cytoskeleton; and (ii) a link to the regulation of cell divisions. The latter aspect is supported by the recent identification of an Arabidopsis mutant with multicellular trichomes, the siamese mutant, suggesting that Arabidopsis trichomes are evolutionarily derived from multicellular forms. We speculate that the spatial information for branch formation is derived from mechanisms employed in cell divisions.     

Dynamic analyses of the expression of the HISTONE::YFP fusion protein in arabidopsis show that syncytial endosperm is divided in mitotic domains

During early seed development, nuclear divisions in the endosperm are not followed by cell division, leading to the development of a syncytium. The simple organization of the Arabidopsis endosperm provides a model in which to study the regulation of the cell cycle in relation to development. To monitor nuclear divisions, we constructed a HISTONE 2B::YELLOW FLUORESCENT PROTEIN gene fusion (H2B::YFP). To validate its use as a vital marker for chromatin in plants, H2B::YFP was expressed constitutively in Arabidopsis. This enabled the observation of mitoses in living root meristems. H2B::YFP was expressed specifically in Arabidopsis syncytial endosperm by using GAL4 transactivation. Monitoring mitotic activity in living syncytial endosperm showed that the syncytium was organized into three domains in which nuclei divide simultaneously with a specific time course. Each mitotic domain has a distinct spatiotemporal pattern of mitotic CYCLIN B1;1 accumulation. The polar spatial organization of the three mitotic domains suggests interactions between developmental mechanisms and the regulation of the cell cycle.     

Potential role of the rice OsCCS52A gene in endoreduplication

In eukaryotes, the cell cycle consists of four distinct phases: G1, S, G2 and M. In certain condition, the cells skip M-phase and undergo endoreduplication. Endoreduplication, occurring during a modified cell cycle, duplicates the entire genome without being followed by M-phase. A cycle of endoreduplication is common in most of the differentiated cells of plant vegetative tissues and it occurs extensively in cereal endosperm cells. Endoreduplication occurs when CDK/Cyclin complex low or inactive caused by ubiquitin-mediated degradation by APC and their activators. In this study, rice cell cycle switch 52 A (OsCCS52A), an APC activator, is functionally characterized using the reverse genetic approach. In rice, OsCCS52A is highly expressed in seedlings, flowers, immature panicles and 15 DAP kernels. Localization studies revealed that OsCCS52A is a nuclear protein. OsCCS52A interacts with OsCdc16 in yeast. In addition, overexpression of OsCCS52A inhibits mitotic cell division and induces endoreduplication and cell elongation in fission yeast. The homozygous mutant exhibits dwarfism and smaller seeds. Further analysis demonstrated that endoreduplication cycles in the endosperm of mutant seeds were disturbed, evidenced by reduced nuclear and cell sizes. Taken together, these results suggest that OsCCS52A is involved in maintaining normal seed size formation by mediating the exit from mitotic cell division to enter the endoreduplication cycles in rice endosperm.     

Reprogramming the Cell Cycle for Endoreduplication in Rodent Trophoblast Cells

Differentiation of trophoblast giant cells in the rodent placenta is accompanied by exit from the mitotic cell cycle and onset of endoreduplication. Commitment to giant cell differentiation is under developmental control, involving down-regulation of Id1 and Id2, concomitant with up-regulation of the basic helix-loop-helix factor Hxt and acquisition of increased adhesiveness. Endoreduplication disrupts the alternation of DNA synthesis and mitosis that maintains euploid DNA content during proliferation. To determine how the mammalian endocycle is regulated, we examined the expression of the cyclins and cyclin-dependent kinases during the transition from replication to endoreduplication in the Rcho-1 rat choriocarcinoma cell line. We cultured these cells under conditions that gave relatively synchronous endoreduplication. This allowed us to study the events that occur during the transition from the mitotic cycle to the first endocycle. With giant cell differentiation, the cells switched cyclin D isoform expression from D3 to D1 and altered several checkpoint functions, acquiring a relative insensitivity to DNA-damaging agents and a coincident serum independence. The initiation of S phase during endocycles appeared to involve cycles of synthesis of cyclins E and A, and termination of S was associated with abrupt loss of cyclin A and E. Both cyclins were absent from gap phase cells, suggesting that their degradation may be necessary to allow reinitiation of the endocycle. The arrest of the mitotic cycle at the onset of endoreduplication was associated with a failure to assemble cyclin B/p34^cdk1 complexes during the first endocycle. In subsequent endocycles, cyclin B expression was suppressed. Together these data suggest several points at which cell cycle regulation could be targeted to shift cells from a mitotic to an endoreduplicative cycle.     

Arabidopsis nucleoporin CPR5 controls trichome cell death through the core cell cycle regulator CKI

• The Arabidopsis trichome is a polyploid epidermal cell resulting from multiple rounds of endocycles. The CYCLIN‐DEPENDENT KINASE INHIBITOR (CKI) family proteins are core cell cycle regulators that promote the endocycle. CONSTITUTIVE EXPRESSION OF PR GENES 5 (CPR5) is a plant‐specific nucleoporin. It has been found that two Arabidopsis CKI, SIAMESE (SIM) and SIAMESE‐RELATED 1 (SMR1), function downstream of CPR5 to activate plant effector‐triggered cell death. The sim smr1 double mutants form multicellular and clustered trichomes, while the cpr5 mutants produce dead and branchless trichomes. This study explored roles of the CPR5‐CKI signalling pathway in trichome cell cycle transition.
• To examine the underlying mechanism of how cell cycle transition is regulated in plant trichomes, Trypan blue staining, flow cytometry, scanning electron microscopy (SEM) and nuclear DNA measurement were conducted.
• The native promoter‐driven CKI and GUS fusion reporter showed that both SIM and SMR1 proteins were preferentially expressed in trichomes. The cpr5‐induced dead and branchless trichomes were fully suppressed by the sim smr1 double mutant, suggesting that SIM and SMR1 function downstream of CPR5 in trichome development. Flow cytometry analysis showed that as compared to the number of 2C (C = DNA content in a haploid nucleus) cells, the number of 4C cells significantly increased, whereas that of polyploidy cells (8C and 16C) dramatically decreased in the cpr5 mutant. The elevated 4C/2C ratio in the cpr5 mutant is consistent with de‐repression of pro‐endocycle regulators SIM and SMR1. The polyploidy cells (8C and 16C) may be selectively targeted to cell death, which is therefore attributed to the branchless trichomes in the cpr5 mutant. Nuclear DNA content analysis demonstrated that the nuclear DNA content of trichomes in the cpr5 sim mutant was significantly higher than in the sim mutant, indicating that CPR5 is a negative endocycle regulator in trichomes.
• This study reveals that the CPR5‐CKI signalling pathway controls trichome cell cycle transition and excessive endocycles are required for cell death in plant trichomes.

     

The Arabidopsis KAKTUS gene encodes a HECT protein and controls the number of endoreduplication cycles

In animals and plants, many cell types switch from mitotic cycles to endoreduplication cycles during differentiation. Little is known about the way in which the number of endoreduplication cycles is controlled in such endopolyploid cells. In this study we have characterized at the molecular level three mutations in the Arabidopsis gene KAKTUS ( KAK), which were previously shown specifically to repress endoreduplication in trichomes. We show that KAK is also involved in the regulation of the number of endoreduplication cycles in various organs that are devoid of trichomes. KAK encodes a protein with sequence similarity to HECT domain proteins. As this class of proteins is known to be involved in ubiquitin-mediated protein degradation, our finding suggests that the number of endoreduplication cycles that occur in several cell types is controlled by this pathway. The KAK gene defines a monophylogenetic subgroup of HECT proteins that also contain Armadillo-like repeats.     

The<Emphasis Type="Italic"> Arabidopsis KAKTUS</Emphasis> gene encodes a HECT protein and controls the number of endoreduplication cycles

In animals and plants, many cell types switch from mitotic cycles to endoreduplication cycles during differentiation. Little is known about the way in which the number of endoreduplication cycles is controlled in such endopolyploid cells. In this study we have characterized at the molecular level three mutations in the Arabidopsis gene KAKTUS ( KAK), which were previously shown specifically to repress endoreduplication in trichomes. We show that KAK is also involved in the regulation of the number of endoreduplication cycles in various organs that are devoid of trichomes. KAK encodes a protein with sequence similarity to HECT domain proteins. As this class of proteins is known to be involved in ubiquitin-mediated protein degradation, our finding suggests that the number of endoreduplication cycles that occur in several cell types is controlled by this pathway. The KAK gene defines a monophylogenetic subgroup of HECT proteins that also contain Armadillo-like repeats.Communicated by G. Jürgens     

An archaebacterial topoisomerase homolog not present in other eukaryotes is indispensable for cell proliferation of plants

Plants, in contrast to other eukaryotes, possess not only homologs of subunit A (AtSPO11-1, 2, 3) but also of subunit B (AtTOP6B) of the archaebacterial topoisomerase VI. AtTOP6B and AtSPO11-3 are strongly expressed in somatic tissue of Arabidopsis and are able to interact with each other in vitro. A T-DNA insertion in AtTOP6B results in deficient cell proliferation; plants stop growing at the rosette stage, have small crinkled leaves, and die about 4 weeks after germination. Cultured root cells die after a limited number of cell divisions. The mitotic index of the root meristems is strongly reduced. Flow cytometric analysis demonstrates that endoreplication in mutant plants is stopped at the 8C stage; the last cycle is not completed in most cases. Mutant plants show a significant increase in nuclear DNA strand breaks. A T-DNA insertion mutant of AtSPO11-3 has a phenotype that is almost to that of AtTOP6B and the double mutant. Thus, both genes seem to act in vivo as subunits of a functional entity. A loss of this function most likely results in a defect in DNA replication, leading directly, or via the activation of a DNA damage checkpoint, to an arrest of cell division and endoreduplication. The dependence on an archaebacterial topoisomerase VI homolog distinguishes plants from the other eukaryotic kingdoms.     

The mitotic inhibitor ccs52 is required for endoreduplication and ploidy-dependent cell enlargement in plants.

Plant organs develop mostly post-embryonically from persistent or newly formed meristems. After cell division arrest, differentiation frequently involves endoreduplication and cell enlargement. Factors controlling transition from mitotic cycles to differentiation programmes have not been identified yet in plants. Here we describe ccs52, a plant homologue of APC activators involved in mitotic cyclin degradation. The ccs52 cDNA clones were isolated from Medicago sativa root nodules, which exhibit the highest degree of endopolyploidy in this plant. ccs52 represents a small multigenic family and appears to be conserved in plants. Overexpression of ccs52 in yeast triggered mitotic cyclin degradation, cell division arrest, endoreduplication and cell enlargement. In Medicago, enhanced expression of ccs52 was found in differentiating cells undergoing endoreduplication. In transgenic M.truncatula plants, overexpression of the ccs52 gene in the antisense orientation resulted in partial suppression of ccs52 expression and decreased the number of endocycles and the volume of the largest cells. Thus, the ccs52 product may switch proliferating cells to differentiation programmes which, in the case of endocycles, result in cell size increments.     

DELLA signaling mediates stress-induced cell differentiation in Arabidopsis leaves through modulation of anaphase-promoting complex/cyclosome activity

Drought is responsible for considerable yield losses in agriculture due to its detrimental effects on growth. Drought responses have been extensively studied, but mostly on the level of complete plants or mature tissues. However, stress responses were shown to be highly tissue and developmental stage specific, and dividing tissues have developed unique mechanisms to respond to stress. Previously, we studied the effects of osmotic stress on dividing leaf cells in Arabidopsis (Arabidopsis thaliana) and found that stress causes early mitotic exit, in which cells end their mitotic division and start endoreduplication earlier. In this study, we analyzed this phenomenon in more detail. Osmotic stress induces changes in gibberellin metabolism, resulting in the stabilization of DELLAs, which are responsible for mitotic exit and earlier onset of endoreduplication. Consequently, this response is absent in mutants with altered gibberellin levels or DELLA activity. Mitotic exit and onset of endoreduplication do not correlate with an up-regulation of known cell cycle inhibitors but are the result of reduced levels of DP-E2F-LIKE1/E2Fe and UV-B-INSENSITIVE4, both inhibitors of the developmental transition from mitosis to endoreduplication by modulating anaphase-promoting complex/cyclosome activity, which are down-regulated rapidly after DELLA stabilization. This work fits into an emerging view of DELLAs as regulators of cell division by regulating the transition to endoreduplication and differentiation.     

In vitro auxin treatment promotes cell division and delays endoreduplication in developing seeds of the model legume species Medicago truncatula

The role of auxins in the morphogenesis of immature seeds of Medicago truncatula was studied, focusing on the transition from the embryo cell division phase to seed maturation. We analyzed seed development in vitro, by flow cytometry, and through the determination of the kinetics of seed fresh weight and size. Thus, seeds were harvested at 8, 10 and 12 days after pollination and cultured in vitro on a medium either without auxin or supplemented with indole‐3‐butyric acid (IBA) or naphthalene acetic acid (NAA) at 1 mg l^?1. All parameters studied were determined every 2 days from the start of in vitro culture. The results showed that both auxins increased the weight and size of seeds with NAA having a stronger effect than IBA. We further demonstrated that the auxin treatments modulate the transition between mitotic cycles and endocycles in M. truncatula developing seed by favoring sustained cell divisions while simultaneously prolonging endoreduplication, which is known to be the cytogenetical imprint of the transition from the cell division phase to the storage protein accumulation phase during seed development.     

The maternal-effect gene futile cycle is essential for pronuclear congression and mitotic spindle assembly in the zebrafish zygote

Embryos have been successfully used for the general study of the cell cycle. Although there are significant differences between the early embryonic and the somatic cell cycle in vertebrates, the existence of specialised factors that play a role during the early cell cycles has remained elusive. We analysed a lethal recessive maternal-effect mutant, futile cycle (fue), isolated in a maternal-effect screen for nuclear division defects in the zebrafish (Danio rerio). The pronuclei fail to congress in zygotes derived from homozygous fue mothers. In addition, a defect in the formation of chromosomal microtubules prevents mitotic spindle assembly and thus chromosome segregation in fue zygotes. However, centrosomal functions do not appear to be affected in fue embryos, suggesting this mutant blocks a subset of microtubule functions. Cleavage occurs normally for several divisions resulting in many anucleate cells, thus showing that nuclear- and cell division can be uncoupled genetically. Therefore, we propose that in mitotic spindle assembly chromosome-dependent microtubule nucleation is essential for the coupling of nuclear and cell division.     

Ectopic Expression of cdc2/cdc28 Kinase Subunit Homo sapiens 1 Uncouples Cyclin B Metabolism from the Mitotic Spindle Cell Cycle Checkpoint

Primary human fibroblasts arrest growth in response to the inhibition of mitosis by mitotic spindle-depolymerizing drugs. We show that the mechanism of mitotic arrest is transient and implicates a decrease in the expression of cdc2/cdc28 kinase subunit Homo sapiens 1 (CKsHs1) and a delay in the metabolism of cyclin B. Primary human fibroblasts infected with a retroviral vector that drives the expression of a mutant p53 protein failed to downregulate CKsHs1 expression, degraded cyclin B despite the absence of chromosomal segregation, and underwent DNA endoreduplication. In addition, ectopic expression of CKsHs1 interfered with the control of cyclin B metabolism by the mitotic spindle cell cycle checkpoint and resulted in a higher tendency to undergo DNA endoreduplication. These results demonstrate that an altered regulation of CKsHs1 and cyclin B in cells that carry mutant p53 undermines the mitotic spindle cell cycle checkpoint and facilitates the development of aneuploidy. These data may contribute to the understanding of the origin of heteroploidy in mutant p53 cells.     

CDC-25.2, a C. elegans ortholog of cdc25, is essential for the progression of intestinal divisions

Intestinal divisions in Caenorhabditis elegans take place in 3 stages: (1) cell divisions during embryogenesis, (2) binucleations at the L1 stage, and (3) endoreduplications at the end of each larval stage. Here, we report that CDC-25.2, a C. elegans ortholog of Cdc25, is required for these specialized division cycles between the 16E cell stage and the onset of endoreduplication. Results of our genetic analyses suggest that CDC-25.2 regulates intestinal cell divisions and binucleations by counteracting WEE-1.3 and by activating the CDK-1/CYB-1 complex. CDC-25.2 activity is then repressed by LIN-23 E3 ubiquitin ligase before the onset of intestinal endoreduplication, and this repression is maintained by LIN-35, the C. elegans ortholog of Retinoblastoma (Rb). These findings indicate that timely regulation of CDC-25.2 activity is essential for the progression of specialized division cycles and development of the C. elegans intestine.