The D-type cyclin CYCD3;1 is limiting for the G1-to-S-phase transition in Arabidopsis

The G1-to-S-phase transition is a key regulatory point in the cell cycle, but the rate-limiting component in plants is unknown. Overexpression of CYCLIN D3;1 (CYCD3;1) in transgenic plants increases mitotic cycles and reduces endocycles, but its effects on cell cycle progression cannot be unambiguously determined. To analyze the cell cycle roles of plant D-type cyclins, we overexpressed CYCD3;1 in Arabidopsis thaliana cell suspension cultures. Changes in cell number and doubling time were insignificant, but cultures exhibited an increased proportion of G2- over G1-phase cells, as well as increased G2 arrest in response to stationary phase and sucrose starvation. Synchronized cultures confirm that CYCD3;1-expressing (but not CYCD2;1-expressing) cells show increased G2-phase length and delayed activation of mitotic genes such as B-type cyclins, suggesting that CYCD3;1 has a specific G1/S role. Analysis of putative cyclin-dependent kinase phosphorylation sites within CYCD3;1 shows that mutating Ser-343 to Ala enhances CYCD3;1 potency without affecting its rate of turnover and results in a fivefold increase in the level of cell death in response to sucrose removal. We conclude that CYCD3;1 dominantly drives the G1/S transition, and in sucrose-depleted cells the decline in CYCD3;1 levels leads to G1 arrest, which is overcome by ectopic CYCD3;1 expression. Ser-343 is likely a key residue in modulating CYCD3;1 activity in response to sucrose depletion.     

D-type cyclins control cell division and developmental rate during Arabidopsis seed development

Seed development in Arabidopsis is characterized by stereotypical division patterns, suggesting that coordinated control of cell cycle may be required for correct patterning and growth of the embryo and endosperm. D-type cyclins (CYCD) are key cell cycle regulators with roles in developmental processes, but knowledge regarding their involvement in seed development remains limited. Here, a family-wide gene expression, and loss- and gain-of-function approach was adopted to reveal additional functions for CYCDs in the development of seed tissues. CYCD genes have both discrete and overlapping tissue-specific expression patterns in the seed as revealed by GUS reporter gene expression. Analysis of different mutant combinations revealed that correct CYCD levels are required in seed development. The CYCD3 subgroup is specifically required as its loss caused delayed development, whereas overexpression in the embryo and endosperm of CYCD3;1 or a previously uncharacterized gene, CYCD7;1, variously leads to induced proliferation, abnormal phenotypes, and elevated seed abortion. CYCD3;1 overexpression provoked a delay in embryonic developmental progression and abnormalities including additional divisions of the hypophysis and suspensor, regions where CYCD3 genes are normally expressed, but did not affect endosperm development. Overexpression of CYCD7;1, not normally expressed in seed development, promoted overgrowth of both embryo and endosperm through increased division and cell enlargement. In contrast to post-germination growth, where pattern and organ size is not generally related to division, results suggest that a close control of cell division through regulation of CYCD activity is important during seed development in conferring both developmental rate and correct patterning.     

Prespore cell fate bias in G1 phase of the cell cycle in Dictyostelium discoideum

By generating a population of Dictyostelium cells that are in the G1 phase of the cell cycle we have examined the influence of cell cycle status on cell fate specification, cell type proportioning and its regulation, and terminal differentiation. The lack of observable mitosis during the development of these cells and the quantification of their cellular DNA content suggests that they remain in G1 throughout development. Furthermore, chromosomal DNA synthesis was not detectable these cells, indicating that no synthesis phase had occurred, although substantial mitochondrial DNA synthesis did occur in prespore cells. The G1-phase cells underwent normal morphological development and sporulation but displayed an elevated prespore/prestalk ratio of 5.7 compared to the 3.0 (or 3:1) ratio normally observed in populations dominated by G2-phase cells. When migrating slugs produced by G1-phase cells were bisected, each half could reestablish the 5.7 (or 5.7:1) prespore/prestalk ratio. These results demonstrate that Dictyostelium cells can carry out the entire developmental cycle in the G1 phase of the cell cycle and that passage from G2 into G1 phase is not required for sporulation. Our results also suggest that the population asymmetry provided by the distribution of cells around the cell cycle at the time of starvation is not strictly required for cell type proportioning. Finally, when developed together with G2-phase cells, G1-phase cells preferentially become prespore cells and exclude G2-phase cells from the prespore-spore cell population, suggesting that G1-phase cells have an advantage over G2-phase cells in executing the spore cell differentiation pathway.     

The Arabidopsis D-type cyclin CYCD4 controls cell division in the stomatal lineage of the hypocotyl epidermis

Cyclin D (CYCD) plays an important role in cell cycle progression and reentry in response to external signals. Here, we demonstrate that Arabidopsis thaliana CYCD4 is associated with specific cell divisions in the hypocotyl. We observed that cycd4 T-DNA insertion mutants had a reduced number of nonprotruding cells and stomata in the hypocotyl epidermis. Conversely, CYCD4 overexpression enhanced cell division in nonprotruding cell files in the upper region of the hypocotyls, where stomata are usually formed in wild-type plants. The overproliferative cells were of stomatal lineage, which is marked by the expression of the TOO MANY MOUTHS gene, but unlike the meristemoids, most of them were not triangular. Although the phytohormone gibberellin promoted stomatal differentiation in the hypocotyl, inhibition of gibberellin biosynthesis did not prevent CYCD4 from inducing cell division. These results suggested that CYCD4 has a specialized function in the proliferation of stomatal lineage progenitors rather than in stomatal differentiation. We propose that CYCD4 controls cell division in the initial step of stomata formation in the hypocotyl.     

A distinct type of cyclin D, CYCD4;2, involved in the activation of cell division in Arabidopsis

The Arabidopsis genome encodes 10 D-type cyclins (CYCD); however, their differential role in cell cycle control is not well known. Among them, CYCD4;2 is unique in the amino acid sequence; namely, it lacks the Rb-binding motif and the PEST sequence that are conserved in CYCDs. Here, we have shown that CYCD4;2 suppressed G1 cyclin mutations in yeast and formed a kinase complex with CDKA;1, an ortholog of yeast Cdc28, in insect cells. Hypocotyl explants of CYCD4;2 over-expressing plants showed faster induction of calli than wild-type explants on a medium containing lower concentration of auxin. These results suggest that CYCD4;2 has a promotive function in cell division by interacting with CDKA;1 regardless of the unusual primary sequence.     

An enhancer trap line associated with a D-class cyclin gene in Arabidopsis

In yeast and animals, cyclins have been demonstrated to be important regulators of cell cycle progression. In recent years, a large number of A-, B-, and D-class cyclins have been isolated from a variety of plant species. One class of cyclins, the D-class cyclins, is important for progression through G1 phase of the cell cycle. In Arabidopsis, four D-class cyclins have been isolated and characterized (CYCLIN-D1;1, CYCLIN-D2;1, CYCLIN-D3;1, and CYCLIN-D4;1). In this report we describe the characterization of a fifth D-class cyclin gene, CYCLIN-D3;2 (CYCD3;2), from Arabidopsis. An enhancer trap line, line 5580, contains a T-DNA insertion in CYCD3;2. Enhancer trap line 5580 exhibits expression in young vegetative and floral primordia. In line 5580, T-DNA is inserted in the first exon of the CYCD3;2 gene; in homozygous 5580 plants CYCD3;2 RNA is not detectable. Even though CYCD3;2 gene function is eliminated, homozygous 5580 plants do not exhibit an obvious growth or developmental phenotype. Via in situ hybridization we demonstrate that CYCD3;2 RNA is expressed in developing vegetative and floral primordia. In addition, CYCD3;2 is also capable of rescuing a yeast strain that is deficient in G1 cyclin activity.     

Expression of genomic AtCYCD2;1 in Arabidopsis induces cell division at smaller cell sizes: implications for the control of plant growth

The Arabidopsis (Arabidopsis thaliana) CYCD2;1 gene introduced in genomic form increased cell formation in the Arabidopsis root apex and leaf, while generating full-length mRNA, raised CDK/CYCLIN enzyme activity, reduced G1-phase duration, and reduced size of cells at S phase and division. Other cell cycle genes, CDKA;1, CYCLIN B;1, and the cDNA form of CYCD2;1 that produced an aberrantly spliced mRNA, produced smaller or zero increases in CDK/CYCLIN activity and did not increase the number of cells formed. Plants with a homozygous single insert of genomic CYCD2;1 grew with normal morphology and without accelerated growth of root or shoot, not providing evidence that cell formation or CYCLIN D2 controls growth of postembryonic vegetative tissues. At the root apex, cells progressed normally from meristem to elongation, but their smaller size enclosed less growth and a 40% reduction in final size of epidermal and cortical cells was seen. Smaller elongated cell size inhibited endoreduplication, indicating a cell size requirement. Leaf cells were also smaller and more numerous during proliferation and epidermal pavement and palisade cells attained 59% and 69% of controls, whereas laminas reached normal size. Autonomous control of expansion was therefore not evident in abundant cell types that formed tissues of root or leaf. Cell size was reduced by a greater number formed in a tissue prior to cell and tissue expansion. Initiation and termination of expansion did not correlate with cell dimension or number and may be determined by tissue-wide signals acting across cellular boundaries.     

Induction of apoptosis and differentiation in neuroblastoma and astrocytoma cells by the overexpression of Bin1, a novel Myc interacting protein.

Bin1 is a novel protein that specifically binds Myc and inhibits, at least in part, Myc transactivation. Bin1 seems to play a role in cell cycle control, acting as a tumor suppressor gene. Since MYC family genes play a regulatory role in the proliferation, differentiation, and apoptosis of the nervous system, we studied the effects of the overexpression of the Myc-interacting protein, Bin1, in neuroblastoma and astrocytoma cell lines, which were chosen as neural cell system models. The major effects of BIN1 overexpression observed in undifferentiated neuroblastoma and astrocytoma cells were a significant reduction of cell growth, an increase in the G(0)/G(1) cell population and the induction of apoptosis. The trigger of programmed cell death by Bin1 is described for the first time. Bin1 overexpression in undifferentiated cells did not induce any maturation process as neither neuronal nor astrocyte differentiation markers were upregulated in neuroblastoma and astrocytoma cells, respectively. On the other side, the effects of Bin1 overproduction in neuroblastoma and astrocytoma cells committed towards neuronal and astrocyte differentiation, respectively, were different from those observed in undifferentiated cells. Although we did not evidence any triggering of programmed cell death, we did notice a further induction towards more differentiated phenotypes. Our studies suggest that Bin1 overexpression in neuroblastoma and astrocytoma cells can result in one of the following pathways: (1) suppressed cell proliferation, (2) induced differentiation, or (3) apoptosis. Thus, it appears that Bin1 operates through different pathways that involve activation of different genes: the chosen pathway however will depend on the proliferating or differentiated state of the cell.     

G1 block of the cell cycle during differentiation of F9 cells correlates with accumulation of inhibitors of the activity of cyclin-kinase complexes of proteins p21/Waf1 and p27/Kip

F9 teratocarcinoma cells have a very short duration of the cell cycle with a short G1-period typical for early embryonic cells. The cells are capable of differentiating towards parietal endoderm cells after the treatment with retinoic acid (RA) and dibutyryl-cAMP (db-cAMP). This leads to changes in the cell cycle; in particular, G1-period becomes longer, and then differentiated F9 cells leave the cycle to stay in G0-phase. It was previously reported that undifferentiated F9 cells undergo no G1 arrest of the cell cycle after DNA damage (Malashicheva et al., 2000). In the present work mechanisms of accumulation of G1-phase cells during differentiation induced by retinoic acid and db-cAMP were studied. Kinase activity of cyclin-Cdk complexes regulating the G1/S transition was analyzed. In differentiated F9 cells, the activity of cyclin-Cdk complexes, comprising Cdk4 and Cdk2 kinases and cyclins A and E, was significantly decreased. A decrease of Cdk4 kinase activity correlates with a drop of the cyclin D1 content. The amount of p21/Waf1 and p27/Kip inhibitors of the cyclin-kinase complexes increased in differentiated F9 cells. p21/Waf1 protein, which undergoes proteasomal degradation in undifferentiated F9 cells, was shown to be stable in their differentiated derivatives. Besides, in differentiated F9 cells p21/Waf1 and p27/Kip proteins can be detected with Cdk4/Cdk2-cyclin E complexes, in contrast to undifferentiated cells. Thus, we suggest that a G1/G0 block of the cell cycle taking place upon differentiation of F9 cells is likely to be caused by a decrease in cyclin-kinase activity due to stabilization and accumulation of p21/Waf1 and p27/Kip inhibitors and to their ability to associate with Cdk-cyclin complexes.     

Brassinosteroids control meristem size by promoting cell cycle progression in Arabidopsis roots

Brassinosteroids (BRs) play crucial roles in plant growth and development. Previous studies have shown that BRs promote cell elongation in vegetative organs in several plant species, but their contribution to meristem homeostasis remains unexplored. Our analyses report that both loss- and gain-of-function BR-related mutants in Arabidopsis thaliana have reduced meristem size, indicating that balanced BR signalling is needed for the optimal root growth. In the BR-insensitive bri1-116 mutant, the expression pattern of the cell division markers CYCB1;1, ICK2/KRP2 and KNOLLE revealed that a decreased mitotic activity accounts for the reduced meristem size; accordingly, this defect could be overcome by the overexpression of CYCD3;1. The activity of the quiescent centre (QC) was low in the short roots of bri1-116, as reported by cell type-specific markers and differentiation phenotypes of distal stem cells. Conversely, plants treated with the most active BR, brassinolide, or mutants with enhanced BR signalling, such as bes1-D, show a premature cell cycle exit that results in early differentiation of meristematic cells, which also negatively influence meristem size and overall root growth. In the stem cell niche, BRs promote the QC renewal and differentiation of distal stem cells. Together, our results provide evidence that BRs play a regulatory role in the control of cell-cycle progression and differentiation in the Arabidopsis root meristem.     

Cyclic AMP-induced differentiated neuroblastoma cells: changes in total nucleic acid and protein contents

The total DNA contents of neuroblastoma cells “differentiated” by dibutyryl cyclic AMP, prostaglandin E₁ and 4-(-3-butoxy-4-methoxybenzyl)-2-imidazolidinone treatment was about 50 percent that of control cells, indicating that cells were accumulated in the G₁-phase of the cell cycle. Sodium butyrate-treated cells were also accumulated in the G₁-phase; however, the expression of “differentiated” phenotype did not occur indicating that inhibition of cell division is not sufficient for morphological differentiation. A marked increase in RNA and protein contents of cyclic AMP-induced “differentiated” cells is consistent with an increase in the size of soma and nucleus.     

Expression pattern of cell cycle genes suggests the developmental arrangement of vascular cells in sugarcane strand

Unlike the ordered multiplication of vascular cells deriving from a row of initials in dicotyledons, vascular growth in monocotyledonous vascular strands does not show the procambial pattern but leads to a complex organization of the vascular bundle. Establishment of the bundle should have a specific developmental pattern. The cell cycle conferring cell proliferation represents a active state of growth and development of tissues. Here, we cloned an A-type CDK gene (Sacof;CDKA;1) from sugarcane (Saccharum officinarum cv. ROC16) and confirmed that its encoding protein interacted physically with two sugarcane CYCD4s (Sacof;CYCD4;1 and Sacof;CYCD4;2), which shared only 47% amino acid sequence similarity. The three genes were expressed concurrently in meristems of root tip, stem tip, and young leaf but not in mature leaves. More importantly, they were predominantly expressed in vascular strands of stem tips and young leaves. In stem-tip strands, the expression region extends deep basipetally to where the sieve tube increases in number in the metaphloem and the vessels are produced in the metaxylem showing a pattern of cell division occurring among differentiating or differentiated cells. This pattern suggests a positional determination of vascular cell arrangement in strands during vascular development.