| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
Plant Physiology 132:2012-2022 (2003) © 2003 American Society of Plant Biologists Constitutive E2F Expression in Tobacco Plants Exhibits Altered Cell Cycle Control and Morphological Change in a Cell Type-Specific MannerPlant Physiology Department, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305–8602, Japan; and Core Research for Evolutional Science and Technology, JST, Ochanomizu, Chiyoda-ku, Tokyo 101–0062, Japan
The E2F family plays a pivotal role in cell cycle control and is conserved among plants and animals, but not in fungi. This provides for the possibility that the E2F family was integrated during the development of higher organisms, but little is known about this. We examined the effect of E2F ectopically expressed in transgenic tobacco (Nicotiana tabacum) plants on growth and development using E2Fa (AtE2F3) and DPa from Arabidopsis. E2Fa-DPa double transgenic lines exhibited altered phenotypes with curled leaves, round shaped petals, and shortened pistils. In mature but not immature leaves of the double transgenic lines, there were enlarged nuclei with increasing ploidy levels accompanied by the ectopic expression of S phase- but not M phase-specific genes. This indicates that a high expression of E2F promotes endoreduplication by accelerating S phase entry in terminally differentiated cells with limited mitotic activity. Furthermore, mature leaves of the transgenic plants contained increased numbers of small cells, especially on the palisade (adaxial) side of the outer region toward the edge, and the leaf strips exhibited hormone-independent callus formation when cultured in vitro. These observations suggest that an enhanced E2F activity modulates cell cycle in a cell type-specific manner and affects plant morphology depending on a balance between activities for committing to S phase and M phase, which likely differ between organs or tissues.
Transition from G1(G0) to S phase is a critical step in the control of the cell cycle, as well as a mitotic step. The control of this process closely involves E2F transcription factors in animals. E2F proteins form heterodimers with DP proteins and regulate promoters of genes involved in deoxynucleotide biosynthesis, DNA replication, and cell cycle control, mainly those required for entering S phase, but also apparently unrelated genes such as the myc and myb families with different expression profiles (Lavia and Jansen-Dürr, 1999  ;
Müller and Helin, 2000).
Recent studies using DNA microarray analysis suggest that E2F regulates also
the expression of genes involved in DNA repair, differentiation, apoptosis,
and mitosis (Ishida et al.,
2001; Müller et al.,
2001; Ren et al.,
2002; Weinmann et al.,
2002).
In humans, six E2F and two DP proteins have been identified. These E2F
members, except for E2F-6, which lacks a transactivation domain, have not only
a transcriptional activating function, but also repressive activity mediated
by the retinoblastoma protein (pRb) or related pocket proteins (p107 and
p130). The E2F/pocket protein complex blocks transcription by masking the
transactivation domain of E2F and by directly binding target promoters to
recruit chromatin-remodeling complexes
(Dyson, 1998
Plants have a conserved regulatory pathway of
G1(G0)-S phase transition mediated by E2F transcription
factors. Promoters or the 5'-untranslated region of proliferating cell
nuclear antigen (PCNA), ribonucleotide reductase (RNR), and MCM3 genes from
tobacco (Nicotiana tabacum), rice (Oryza sativa), and
Arabidopsis contain E2F sites that are responsible for the S phase or
meristematic tissue-specific expression and that bind E2F/DP complexes from
plants (Chaboute et al., 2000
It is quite important to understand how the control of
G1(G0)-S transition by the E2F family affects plant
growth, proliferation, and development. Although the functions of these plant
E2Fs, as well as animal E2Fs, have been well studied in cultured cells, little
is known about the role of E2F in whole organisms. In mammalian cells and
imaginal discs of transgenic flies, overexpression of E2F induces S-phase
entry followed by apoptosis. In many cases, the E2F-induced apoptosis
abolishes the next round of cell cycle progression
(Qin et al., 1994 We analyzed the effect of E2Fa (AtE2F3) and DPa on the regulation of the cell cycle and development using transgenic tobacco plants overexpressing these proteins. In contrast to the transgenic Arabidopsis plants, E2Fa-DPa double transgenic tobacco plants grew until a late stage and exhibited morphological abnormalities specific to certain organs. The ectopic expression of E2Fa and DPa induced not only endoreduplication and/or cell division, which were specifically enhanced in specific tissues of mature leaves, but also hormone-independent formation of callus in leaf strips cultured in vitro. These observations suggest that the activity of E2F can induce S phase entry and continuous cell cycle progression or cell cycle arrest, thereby modulating tissue and organ growth in the presence or absence of other cell cycle-associated factors such as mitotic factors.
Phenotype of Tobacco Plants Overexpressing E2Fa and DPa
We have previously demonstrated that E2Fa (AtE2F3) exerts its
transactivation function when DPa is coexpressed in cultured tobacco cells
(Kosugi and Ohashi, 2002c Two lines of E2Fa-overexpressing plants with a high and low level of expression were crossed with a DPa-overexpressing plant. Seedlings of both crossed lines (designated H-F3D and L-F3D, respectively, for high and low levels of transgene expression) had small and downward curled cotyledons (data not shown). The H-F3D line exhibited severely delayed growth and almost all the plants died when 15 to 20 cm in height before or during flowering. All leaves of this line curled downward, and at later stages, developed chlorosis and spontaneous lesions reminiscent of the symptoms observed on a geminivirus-infected tobacco plant (Fig. 1, B and C). In contrast, the L-F3D line grew relatively normally in the early stages, but formed curled leaves similar to those of H-F3D plants at the adult stage (Fig. 1D). Other interesting phenotypes were observed in the flowers of both lines: petals of a rounder shape and pistils of a shorter length than wild-type flowers (Fig. 1, E and F).
To confirm whether the phenotypical disorders in these crossed lines are linked to the expression levels of both transgenes, we performed reverse transcriptase (RT)-PCR analysis with primers specific for the E2Fa and DPa cDNAs, and a tobacco PCNA gene, an endogenous E2F target in tobacco. Whereas phenotypically normal leaves from L-F3D expressed low levels of the E2Fa and DPa genes, the deformed leaves from this line expressed high levels of both transgenes, comparable with the H-F3D line (Fig. 2A). The RT-PCR analysis further demonstrated that the endogenous PCNA gene was more active in the phenotypically altered leaves of both crossed lines. These results suggest that the transgene expression in the L-F3D line is developmentally regulated probably via an epigenetic effect by which the transgenes are reactivated at a later stage, and furthermore, that the phenotypical disorders observed are caused by the overexpression of E2Fa and DPa.
The functional expression of the transgenes was further confirmed by EMSA
with nuclear extracts prepared from mature leaves of the crossed and the
original single transgenic lines. Nuclear extracts from the H-F3D line
exhibited a high level of activity for binding to te2f-1 and te2f-
To test whether in E2Fa-DPa transgenic plants, the expression of endogenous target genes other than the PCNA gene is affected, several tobacco genes, including cell cycle-regulated genes, were analyzed by northern-blot analysis. Fully expanded mature leaves of the control 35S-GUS and single transgenic plants showed no detectable or only a low level of expression of cell cycle-regulated genes; S phase-specific genes associated with DNA synthesis (PCNA and RNR) and chromosome assembly (histone H1 and histone H3), and M phase-specific genes (B-type cyclins; Fig. 3). Only the E2Fa-DPa double transgenic line (H-F3D) expressed ectopically high levels of DNA synthesis genes and moderate levels of histone genes, but not M phase and other cell cycle-unrelated genes involved in glycolysis (GAPDH) and photosynthesis (Cab). Also in immature leaves ranging from 10 to 15 mm, the plant expressed higher levels of these S phase genes than the control and other single E2Fa or DPa transgenic lines. In lines expressing low levels of E2Fa-DPa (L-F3D), moderate levels of PCNA transcripts were expressed in mature leaves, and the levels of B-type cyclin transcripts were similar to those of the control plants in mature and immature leaves (data not shown).
It is not clear whether the S phase genes such as histones H1 and H3 are
direct targets for E2F in plants, although histone genes have been observed to
be regulated directly by E2F in mammalian cells
(Yagi et al., 1995
A number of cell cycle-regulated genes in plants are strictly expressed in
the cell cycle-dependent manner in meristematic tissues, as evident from the
patch-like pattern of expression in shoot apical meristematic regions
(Fobert et al., 1994
To assess whether the ectopic S phase entry induced by E2Fa/DPa confers an ectopic cell division or endoreplication in the transgenic plants, we observed microscopically cell morphology in mature and immature leaves. Cells in immature leaves of the E2Fa-DPa (H-F3D and L-F3D) and the control 35S-GUS plants were similar in size and number (Fig. 5, G–I). In contrast, mature leaves of these two E2F transgenic lines contained smaller and increased numbers of cells than the control plants, despite that the leaf size was similar between them (Fig. 5, A–C). The smaller cell size of both transgenic lines was more striking in the palisade tissues of the outer region of the leaves, especially at the edge (data not shown). This observation suggests that the E2Fa-DPa expression stimulates cell division in a cell type-specific manner.
Interestingly, the spongy tissues of the transgenic lines had an increased
number of idioblastic cells (Fig. 5,
A–C). Crystal idioblasts deposited with calcium oxalate
crystals, appearing dark under a light microscope, have been observed in
various tissues and plant species (for review, see
Franceschi, 2001 To observe the nuclear morphology, these leaf sections were stained with DAPI. In immature leaves of the H-F3D plants, the nuclei were indistinguishable in size from those of the control 35S-GUS plants (Fig. 5, G–I). Also, nuclei in meristematic regions of shoot and root apical tips were similar in size in these plants (data not shown). In contrast, mature leaves of the H-F3D plants contained considerably larger nuclei than the 35S-GUS plants (Fig. 5, D and E). The enlarged nuclei were more abundant in cells in the spongy tissue and epidermal cells, including trichomes, than in the palisade tissue, suggesting the presence of tissue specificity in the E2Fa/DPa-activated endoreplication. In the L-F3D plants, nuclear size, unlike cellular size, in the mature leaves was apparently similar to the control (Fig. 5F). We then measured the nuclear DNA content in the mature leaves. The amount of genomic DNA isolated from the E2Fa-DPa H-F3D line was approximately 8-fold that of the 35S-GUS plants and the E2Fa or DPa single transgenic line (Fig. 6A). We also measured ploidy levels in the mesophyll cells by flow cytometric analysis. Almost all nuclei in mature leaves of the 35S-GUS plant had a ploidy level of 2C. In contrast, the L-F3D line exhibited an increased number of nuclei with a ploidy level of 4C, and the H-F3D line had even higher ploidy levels, varying from 2C to 32C, with the most abundant population having 4C (Fig. 6B). These results indicate that the ectopic E2Fa-DPa expression causes endoreduplication by stimulating DNA synthesis in terminally differentiated tissues.
We examined the effect of plant hormones stimulating cell growth and
division on in vitro cultures of leaf strips from E2Fa-DPa plants. Leaf strips
from the E2Fa or DPa single transgenic line induced regeneration of roots,
callus, or shoots on a medium containing
The calli of the H-F3D line stopped showing cell proliferation at an early stage, while 1 to 3 mm in size, whereas calli of the L-F3D line continued to grow (Fig. 7B). To examine the difference in the growth competence of the calli of both lines of transgenic plants, RT-PCR was conducted to detect the expression of S and M phase-specific genes in the in vitro cultured leaf strips. The PCNA transcript was expressed not only in leaf strips of both lines of transgenic plants, but also in the control 35S-GUS plant when the strips were cultured with the hormone-free medium for 24 h (Fig. 7C). Although B-type cyclin transcripts were also produced on culturing the leaf strips of the 35S-GUS and L-F3D plants, leaf strips from the H-F3D line had fewer transcripts of the cyclin genes (Fig. 7C). These results suggest that the growth arrest of the H-F3D calli is due to impaired induction of the B-type cyclin genes in the in vitro culture system.
Morphological Change Caused by Ectopic E2F-DP Expression in Plants
Our previous analyses have demonstrated that Arabidopsis expresses two DP
(DPa and DPb) and three E2F (E2Fa-c/AtE2F1-3) homologs with an overall
similarity to animal E2Fs. E2Fa and E2Fb acquire the function as potent
transcriptional activators by stimulating DNA binding and nuclear import upon
interacting with DPa (Kosugi and Ohashi,
2002c
The phenotype accompanying leaf curving and chlorosis observed in the H-F3D
line is similar to that observed in tobacco (Nicotiana benthamiana)
plants infected with geminiviruses such as tomato golden mosaic virus (TGMV)
and squash leaf curl virus (von Arnim and
Stanley, 1992 Unlike the E2Fa-DPa transgenic Arabidopsis, all of the L-F3D and a few of the H-F3D tobacco plants continued to grow until the flowering stage. All flowers produced in the L-F3D plants displayed phenotypes of shorter pistils and rounder-shaped petals than wild-type flowers, despite that the CaMV 35S promoter directs a ubiquitous expression and phenotypes of other organs such as the size of petals and anthers were less affected. These observations suggest that the activity for driving S phase entry by E2Fa and DPa can modulate organ size or morphology by regulating growth of the organs. The organ and tissue specificity of the morphological change may be determined by other factors modulating the E2Fa/DPa activity or by mitotic factors, which are abundant in the organs showing altered phenotypes. If the E2F activity or modulating effects are distributed differently among tissues or species, or in different environments, plant architecture could be at least partly responsible for the control of cell proliferation mediated by E2F activity.
In animals, overexpression of E2F induces S-phase entry followed by
apoptosis (Johnson et al.,
1993
On the other hand, there was an increase in the number of small cells in
the mesophyll tissue of the E2Fa-DPa plants, despite that the size and
developmental stage of the leaves observed was similar between the control and
transgenic plants. This indicates that the enhanced E2F activity induces
ectopic cell division in tobacco. It has been shown that E2Fa-DPa transgenic
Arabidopsis lines display ectopic cell division in the cotyledons and
hypocotyls (De Veylder et al.,
2002 In the leaves of E2Fa-DPa tobacco plants, there was a greater increase in the number of cells on the palisade side in the regions toward the edge (outer region), indicating that cell proliferation is stimulated more in the outer palisade tissue than in the inner region and spongy tissue. Taken together with the fact that the enlargement of nuclei is remarkable in spongy tissue of H-F3D plants, mitotic activity on the abaxial side is likely to be more limited than that on the adaxial side in the leaf. These observations further suggest that the phenotype of leaf curling is caused by an accelerated proliferation of adaxial mesophyll cells in the outer region and by repressed cell division on the abaxial side.
The E2Fa-DPa double transgenic plants had the ability to form callus when their leaf sections were cultured with a hormone-free medium. Our study using RT-PCR analyses demonstrates that the culturing of leaf sections in the absence of hormones, probably in combination with the wounding effect, induces the re-entry of terminally differentiated cells into the cell cycle, as shown by the elevated expression of the PCNA and B-type cyclin transcripts in the cultured leaf strips. Leaf sections from wild-type tobacco incidentally formed small calli when cultured in the absence of hormones, but there was no further callus growth (data not shown). These observations suggest that the ectopic expression of E2Fa and DPa stimulates the growth of calli but not the initiation. We speculate that once a callus has formed on cultured leaf strips, mitotic activity is constantly provided, but activity for S phase entry is not sufficient to stimulate further growth of the callus. Calli produced in the H-F3D line stopped growing at an early stage, apparently in correlation with the impaired expression of B-type cyclin transcripts in the cultured H-F3D leaves. Considering that the H-H3D plants exhibited delayed growth and a lower degree of ectopic cell division than the L-F3D plants, a higher level of E2Fa-DPa expression may inhibit expression of mitotic genes.
Our observations suggest that a high level of E2F activity induces S phase
entry and enhances endoreduplication in terminally differentiated tissues, and
that additional mitotic activity to enter M phase could stimulate continuous
cell cycle progression. It has been reported that the overexpression of
Arabidopsis D-type cyclin species stimulates growth in tobacco plants
(Cockcroft et al., 2000
Plasmid Construction
For plant transformation constructs, XbaI-XhoI fragments
from the E2Fa (AtE2F3) and DPa cDNA clones
(Kosugi and Ohashi, 2002c
The binary vectors were introduced into Agrobacterium tumefaciens
LBA4404 and were used for transformation of tobacco (Nicotiana
tabacum) as described previously
(Kosugi et al., 1991
First strand cDNA was synthesized with 5 µg of total RNA, as described
previously (Kosugi and Ohashi,
2002b
The preparation of nuclear extracts from tobacco leaves and EMSAs were
performed as previously described (Kosugi
and Ohashi, 1997
Total RNA was isolated from mature and immature leaves of tobacco with
Trizol reagent, according to the manufacturer's instructions (Invitrogen,
Carlsbad, CA). RNA (20 µg) was fractionated in 1% (w/v)
formaldehyde-agarose gels and was transferred to a nylon filter (Hybond
N+; Amersham Biosciences, Piscataway, NJ) as described
(Sambrook et al., 1989
Shoot tips from 35S-GUS and 35S-E2Fa-DPa plants were fixed with 4% (w/v)
paraformaldehyde. The preparation of paraffin sections and in situ detection
using DIG-labeled RNA probe were conducted as described
(Kouchi et al., 1995
Strips of mature and immature leaves from 35S-GUS and 35S-E2Fa-DPa transgenic plants were embedded in 5% (w/v) agar and sliced into 60-µm thick cross-sections using a microslicer (DTK-1000; Dohan-EM, Kyoto). The sections were incubated with 20% (w/v) ethanol containing 1 µg mL–1 DAPI for 5 min. Nuclei were observed using an epifluorescent microscope (model AX70; Olympus, Tokyo).
Nuclear DNA was isolated from 200 mg of mature leaves with a Nucleon PhytoPure kit according to the manufacturer's instructions (Amersham Biosciences). The isolated DNA was quantified using a spectrophotomer and simultaneously by a fluorometrical method with DAPI, and was normalized relative to the protein content of the leaves sampled.
Leaf strips from 35S-GUS and 35S-E2Fa-DPa plants were chopped in
Galbraith's buffer (Galbraith et al.,
1983
We thank Dr. Tetsuji Kakutani for advice on the culture, transformation, and crossing of Arabidopsis plants. We are grateful to Drs. Akemi Tagiri and Taka Murakami for tips on sectioning of tissues and in situ hybridization technique. Received April 8, 2003; returned for revision May 6, 2003; accepted May 12, 2003.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.025080.
1 Present address: Institute for Advanced Biosciences, Keio University,
Daihoji Tsuruoka, Yamagata 997–0017, Japan. * Corresponding author; e-mail yohashi{at}affrc.go.jp; fax 81–298–38–7469.
Albani D, Mariconti L, Ricagno S, Pitto L, Moroni C, Helin K, Cella R (2000) DcE2F, a functional plant E2F-like transcriptional activator from Daucus carota. J Biol Chem 275: 19258–19267
Asano M, Nevins JR, Wharton RP (1996) Ectopic
E2F expression induces S phase and apoptosis in Drosophila imaginal
discs. Genes Dev 10:
1422–1432
Chaboute ME, Clement B, Philipps G (2002) S
phase and meristem-specific expression of the tobacco RNR1b gene is mediated
by an E2F element located in the 5' leader sequence. J Biol
Chem 277:
17845–17851
Chaboute ME, Clement B, Sekine M, Philipps G, Chaubet-Gigot
N (2000) Cell cycle regulation of the tobacco ribonucleotide
reductase small subunit gene is mediated by E2F-like elements. Plant
Cell 12:
1987–2000 Cockcroft CE, den Boer BG, Healy JM, Murray JA (2000) Cyclin D control of growth rate in plants. Nature 405: 575–579[CrossRef][Medline] DeGregori J (2002) The genetics of the E2F family of transcription factors: shared functions and unique roles. Biochim Biophys Acta 1602: 131–150[Medline] de Jager SM, Menges M, Bauer UM, Murra JA (2001) Arabidopsis E2F1 binds a sequence present in the promoter of S-phase-regulated gene AtCDC6 and is a member of a multigene family with differential activities. Plant Mol Biol 47: 555–568[CrossRef][ISI][Medline]
Del Pozo JC, Boniotti MB, Gutierrez C (2002)
Arabidopsis E2Fc functions in cell division and is degraded by the
ubiquitin-SCF(AtSKP2) pathway in response to light. Plant Cell
14:
3057–3071 De Veylder L, Beeckman T, Beemster GT, de Almeida Engler J, Ormenese S, Maes S, Naudts M, Van Der Schueren E, Jacqmard A et al. (2002) Control of proliferation, endoreduplication and differentiation by the Arabidopsis E2Fa-DPa transcription factor. EMBO J 21: 1360–1368[CrossRef][ISI][Medline] Du W, Xie JE, Dyson N (1996) Ectopic expression of dE2F and dDP induces cell proliferation and death in the Drosophila eye. EMBO J 15: 3684–3692[Medline]
Dyson N (1998) The regulation of E2F by
pRB-family proteins. Genes Dev
12:
2245–2262 Edgar BA, Orr-Weaver TL (2001) Endoreplication cell cycles: more for less. Cell 105: 297–306[CrossRef][ISI][Medline]
Egelkrout EM, Mariconti L, Settlage SB, Cella R, Robertson D,
Hanley-Bowdoin L (2002) Two E2F elements regulate the
proliferating cell nuclear antigen promoter differently during leaf
development. Plant Cell 14:
3225–3236
Egelkrout EM, Robertson D, Hanley-Bowdoin L
(2001) Proliferating cell nuclear antigen transcription is
repressed through an E2F consensus element and activated by geminivirus
infection in mature leaves. Plant Cell
13:
1437–1452 Fobert PR, Coen ES, Murphy GJ, Doonan JH (1994) Patterns of cell division revealed by transcriptional regulation of genes during the cell cycle in plants. EMBO J 13: 616–624[ISI][Medline] Franceschi V (2001) Calcium oxalate in plants. Trends Plant Sci 6: 331[Medline]
Galbraith DWHK, Maddox JM, Ayres NM, Sharma DP, Firoozabadi
E (1983) Rapid flow cytometric analysis of the cell cycle in
intact plant tissues. Science
220:
1049–1051 Gaubatz S, Lindeman GJ, Ishida S, Jakoi L, Nevins JR, Livingston DM, Rempel RE (2000) E2F4 and E2F5 play an essential role in pocket protein-mediated G1 control. Mol Cell 6: 729–735[CrossRef][ISI][Medline]
Ishida S, Huang E, Zuzan H, Spang R, Leone G, West M, Nevins
JR (2001) Role for E2F in control of both DNA replication and
mitotic functions as revealed from DNA microarray analysis. Mol Cell
Biol 21:
4684–4699 Johnson DG, Schwarz JK, Cress WD, Nevins JR (1993) Expression of transcription factor E2F1 induces quiescent cells to enter S phase. Nature 365: 349–352[CrossRef][Medline] Joubes J, Chevalier C (2000) Endoreduplication in higher plants. Plant Mol Biol 43: 735–745[CrossRef][ISI][Medline] Kong LJ, Orozco BM, Roe JL, Nagar S, Ou S, Feiler HS, Durfee T, Miller AB, Gruissem W, Robertson D et al. (2000) A geminivirus replication protein interacts with the retinoblastoma protein through a novel domain to determine symptoms and tissue specificity of infection in plants. EMBO J 19: 3485–3495[CrossRef][ISI][Medline] Kosugi S, Ohashi Y (1997) PCF1 and PCF2 specifically bind to cis elements in the rice proliferating cell nuclear antigen gene. Plant Cell 9: 1607–1619[Abstract]
Kosugi S, Ohashi Y (2000) Cloning and
DNA-binding properties of a tobacco ethylene-insensitive3 (EIN3) homolog.
Nucleic Acids Res 28:
960–967 Kosugi S, Ohashi Y (2002a) E2F sites that can interact with E2F proteins cloned from rice are required for meristematic tissue-specific expression of rice and tobacco proliferating cell nuclear antigen promoters. Plant J 29: 45–59[CrossRef][ISI][Medline]
Kosugi S, Ohashi Y (2002b) E2Ls, E2F-like
repressors of Arabidopsis that bind to E2F sites in a monomeric form. J
Biol Chem 277:
16553–16558
Kosugi S, Ohashi Y (2002c) Interaction of the
Arabidopsis E2F and DP proteins confers their concomitant nuclear
translocation and transactivation. Plant Physiol
128:
833–843
Kosugi S, Suzuka I, Ohashi Y, Murakami T, Arai Y
(1991) Upstream sequences of rice proliferating cell nuclear
antigen (PCNA) gene mediate expression of PCNA-GUS chimeric gene in meristems
of transgenic tobacco plants. Nucleic Acids Res
19:
1571–1576 Kouchi H, Sekine M, Hata S (1995) Distinct classes of mitotic cyclins are differentially expressed in the soybean shoot apex during the cell cycle. Plant Cell 7: 1143–1155[Abstract] Lavia P, Jansen-Dürr P (1999) E2F target genes and cell-cycle checkpoint control. Bioessays 21: 221–230[CrossRef][ISI][Medline]
Lindeman GJ, Dagnino L, Gaubatz S, Xu Y, Bronson RT, Warren
HB, Livingston DM (1998) A specific, nonproliferative
role for E2F-5 in choroid plexus function revealed by gene targeting.
Genes Dev 12:
1092–1098 Magyar Z, Atanassova A, De Veylder L, Rombauts S, Inzé D (2000) Characterization of two distinct DP-related genes from Arabidopsis thaliana. FEBS Lett 486: 79–87[CrossRef][ISI][Medline]
Mariconti L, Pellegrini B, Cantoni R, Stevens R, Bergounioux C,
Cella R, Albani D (2002) The E2F family of
transcription factors from Arabidopsis thaliana: novel and conserved
components of the retinoblastoma/E2F pathway in plants. J Biol
Chem 277:
9911–9919
Müller H, Bracken AP, Vernell R, Moroni MC, Christians F,
Grassilli E, Prosperini E, Vigo E, Oliner JD, Helin K
(2001) E2Fs regulate the expression of genes involved in
differentiation, development, proliferation, and apoptosis. Genes
Dev 15:
267–285 Müller H, Helin K (2000) The E2F transcription factors: key regulators of cell proliferation. Biochim Biophys Acta 1470: M1–12[Medline] Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15: 473–497[CrossRef] Nagar S, Pedersen TJ, Carrick KM, Hanley-Bowdoin L, Robertson D (1995) A geminivirus induces expression of a host DNA synthesis protein in terminally differentiated plant cells. Plant Cell 7: 705–719[Abstract]
Ogawa H, Ishiguro K, Gaubatz S, Livingston DM, Nakatani Y
(2002) A complex with chromatin modifiers that occupies E2F- and
Myc-responsive genes in G0 cells. Science
296:
1132–1136
Paramio JM, Segrelles C, Casanova ML, Jorcano JL
(2000) Opposite functions for E2F1 and E2F4 in human epidermal
keratinocyte differentiation. J Biol Chem
275:
41219–41226
Pascal E, Goodlove PE, Wu LC, Lazarowitz SG
(1993) Transgenic tobacco plants expressing the geminivirus BL1
protein exhibit symptoms of viral disease. Plant Cell
5:
795–807
Qin XQ, Livingston DM, Kaelin WG Jr, Adams PD
(1994) Deregulated transcription factor E2F-1 expression leads to
S-phase entry and p53-mediated apoptosis. Proc Natl Acad Sci
USA 91:
10918–10922 Ramírez-Parra E, Gutierrez C (2000) Characterization of wheat DP, a heterodimerization partner of the plant E2F transcription factor which stimulates E2F-DNA binding. FEBS Lett 486: 73–78[CrossRef][ISI][Medline]
Ramírez-Parra E, Xie Q, Boniotti MB, Gutierrez C
(1999) The cloning of plant E2F, a retinoblastoma-binding
protein, reveals unique and conserved features with animal G(1)/S regulators.
Nucleic Acids Res 27:
3527–3533
Ren B, Cam H, Takahashi Y, Volkert T, Terragni J, Young RA,
Dynlacht BD (2002) E2F integrates cell cycle
progression with DNA repair, replication, and G(2)/M checkpoints. Genes
Dev 16:
245–256
Riou-Khamlichi C, Huntley R, Jacqmard A, Murray JA
(1999) Cytokinin activation of Arabidopsis cell division through
a D-type cyclin. Science 283:
1541–1544 Rossignol P, Stevens R, Perennes C, Jasinki S, Cella R, Tremousaygue D, Bergounioux C (2002) AtE2F-a and AtDP-a, members of the E2F family of transcription factors, induce Arabidopsis leaf cells to re-enter S phase. Mol Genet Genomics 266: 995–1003[CrossRef][Medline] Sambrook J, Fritsch EF, and Maniatis T (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
Schnittger A, Schobinger U, Bouyer D, Weinl C, Stierhof YD,
Hulskamp M (2002a) Ectopic D-type cyclin expression
induces not only DNA replication but also cell division in Arabidopsis
trichomes. Proc Natl Acad Sci USA
99:
6410–6415 Schnittger A, Schobinger U, Stierhof YD, Hulskamp M (2002b) Ectopic B-type cyclin expression induces mitotic cycles in endoreduplicating Arabidopsis trichomes. Curr Biol 12: 415–420[CrossRef][ISI][Medline] Sekine M, Ito M, Uemukai K, Maeda Y, Nakagami H, Shinmyo A (1999) Isolation and characterization of the E2F-like gene in plants. FEBS Lett 460: 117–122[CrossRef][ISI][Medline]
Shan B, Lee WH (1994) Deregulated expression of
E2F-1 induces S-phase entry and leads to apoptosis. Mol Cell
Biol 14:
8166–8173
Stevens R, Mariconti L, Rossignol P, Perennes C, Cella R,
Bergounioux C (2002) Two E2F sites in the Arabidopsis MCM3
promoter have different roles in cell cycle activation and meristematic
expression. J Biol Chem 277:
32978–32984
Trimarchi JM, Fairchild B, Wen J, Lees JA
(2001) The E2F6 transcription factor is a component of the
mammalian Bmi1-containing polycomb complex. Proc Natl Acad Sci
USA 98:
1519–1524
Vandepoele K, Raes J, De Veylder L, Rouzé P, Rombauts S,
Inzé D (2002) Genome-wide analysis of core cell cycle
genes in Arabidopsis. Plant Cell
14:
903–916 von Arnim A, Stanley J (1992) Determinants of tomato golden mosaic virus symptom development located on DNA B. Virology 186: 286–293[CrossRef][Medline]
Wang D, Russell JL, Johnson DG (2000) E2F4 and
E2F1 have similar proliferative properties but different apoptotic and
oncogenic properties in vivo. Mol Cell Biol
20:
3417–3424
Weinmann AS, Yan PS, Oberley MJ, Huang TH, Farnham PJ
(2002) Isolating human transcription factor targets by coupling
chromatin immunoprecipitation and CpG island microarray analysis. Genes
Dev 16:
235–244 Wu L, Timmers C, Maiti B, Saavedra HI, Sang L, Chong GT, Nuckolls F, Giangrande P, Wright FA, Field SJ et al. (2001) The E2F1–3 transcription factors are essential for cellular proliferation. Nature 414: 457–462[CrossRef][Medline]
Yagi H, Kato T, Nagata T, Habu T, Nozaki M, Matsushiro A,
Nishimune Y, Morita T (1995) Regulation of the mouse
histone H2A: X gene promoter by the transcription factor E2F and CCAAT binding
protein. J Biol Chem 270:
18759–18765 This article has been cited by other articles:
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
TOP
ABSTRACT
RESULTS
-glucuronidase (GUS) plant as a control. B, Nine-week-old
35S-E2Fa-DPa plant (H-F3D line) with a high level of expression of the
transgenes. Leaves are partially curled downward and are rounder relative to
the control plant. C, Sixteen-week-old H-F3D line. Leaves, especially upper
leaves, develop chlorosis and spontaneous lesions. D, L-F3D line of
35S-E2Fa-DPa transgenics. Leaves are partially curled. The left shoot, which
was formed at a later growth stage, has leaves with an extremely curled
surface. E, Phenotype of flowers of a control 35S-GUS (left) and an L-F3D
(right) plant. The round shape of the carpel of the L-F3D line contrasts with
the sharp features of a control flower. F, Inner architecture of 35S-GUS
(left) and L-F3D (right) flowers. Arrowheads indicate positions of stigma. The
L-F3D line has a considerably shorter pistil than the control.:H-F3D and
L-F3D, Lines of E2Fa-DPa double transformants with a high and low level of
expression of the E2Fa transgene, respectively.
contain different E2F-binding sequences and mte2f-1
contains a mutated sequence of te2f-1. The arrowhead indicates the position of
a complex of E2Fa/DPa with the probe DNA.
