Control of the Endocycle by Cell Cycle Related Genes

Endoreduplication is often viewed as a short-cut of the mitotic cell cycle that skips mitosis and re-enters the S-phase. During the normal mitotic cell cycle, cells have a mechanism to prevent entry into the S-phase without going through the M-phase. Cells that initiate the endocycle, however, are able to break this rule and re-enter the S phase in the absence of mitosis (Fig. 3). Despite these key differences between the mitotic cell cycle and endocycle, at least some of the molecular machineries that trigger the S-phase appear to be commonly shared between these two types of cell cycles and only subsets of these controls are specific to the endocycle (Table 1). E2F is a transcription factor that regulates entry into the S-phase during the mitotic cell cycle. E2F

Growth Related Genes Plants

Fig. 3 A schematic diagram that describes how endoreduplication may be controlled in plants. During the endocycle, cells re-enter the S-phase without going through the M-phase. Whether there is a gap phase (G phase) between successive S-phases is not currently known. A key step to switch from the mitotic cell cycle to endocycle is to drop the M-phase-specific CDK activity to inhibit the induction of mitosis and this process is mediated through the down-regulation of M-phase-specific cyclins such as CYCAs and CYCBs. Re-entry into the S-phase during the endocycle utilizes several E2F transcription factors E2Fa, E2Fc and DEL1/E2Fe that also regulate S-phase entry in the mitotic cell cycle. DNA replication during the endocycle uses mostly the same molecular machinery as that used for the S-phase in the mitotic cell cycle, e.g. components of a pre-replication complex such as CDC6 and CDT1, but at least some including components of the plant DNA topoisomerase VI, SPO11-3, TOP6B and RHL1, appear to have a specific role in the endocycle

Fig. 3 A schematic diagram that describes how endoreduplication may be controlled in plants. During the endocycle, cells re-enter the S-phase without going through the M-phase. Whether there is a gap phase (G phase) between successive S-phases is not currently known. A key step to switch from the mitotic cell cycle to endocycle is to drop the M-phase-specific CDK activity to inhibit the induction of mitosis and this process is mediated through the down-regulation of M-phase-specific cyclins such as CYCAs and CYCBs. Re-entry into the S-phase during the endocycle utilizes several E2F transcription factors E2Fa, E2Fc and DEL1/E2Fe that also regulate S-phase entry in the mitotic cell cycle. DNA replication during the endocycle uses mostly the same molecular machinery as that used for the S-phase in the mitotic cell cycle, e.g. components of a pre-replication complex such as CDC6 and CDT1, but at least some including components of the plant DNA topoisomerase VI, SPO11-3, TOP6B and RHL1, appear to have a specific role in the endocycle

Table 1 Mutants and transgenic plants that show ploidy phenotypes

Gene Expected function General phenotypes Ploidy phenotypes Refs.

(A) Genes identified from gain-of-function studies Cell cycle signalling

E2Fa Gl/S transition, Dwarf1

and DPa transcription factor

CDC6a Component of DNA replication Over-branched trichomes, licensing complex increased stomatal density1

CDTla Component of DNA replication Over-branched trichomes,

CYCB1;2 ICK1/KRP1

licensing complex M cyclin CDK inhibitor

ICK2/KRP2 CDK inhibitor

ILP1

Transcriptional repressor

Light signalling

IPD1 CUE domain protein increased stomatal density1 Multicellular trichomes2 Small/under-branched trichomes2

Small and serrated leaves, large leaf cells1 Thick hypocotyls, large cotyledons, increased cell size in seedlings3

Slightly longer hypocotyls in the dark3

(B) Genes identified from loss-of-function studies Cell cycle signalling

E2Fc Gl/S transition, Small curled leaves, transcription factor reduced cell size4

DELI DP-E2F-like, Not known5

transcription factor

Increased in seedlings and trichomes Increased in leaves and trichomes Increased in leaves and trichomes Reduced in trichomes Reduced in trichomes, increased in neighboring cells Reduced in leaves

Increased in seedlings and leaves

Increased in dark-grown hypocotyls

Reduced in leaves

Increased in seedlings and true leaves

De Veylder et al. 2002

Castellano et al. 2001, 2004

Castellano et al. 2004

Schnittger et al. 2003 Schnittger et al. 2003; Weinl et al. 2005 De Veylder et al. 2001

Yoshizumi et al. 2006

Tsumoto et al. 2006

del Pozo et al. 2006 Vlieghe et al. 2005

Gene

Expected function

General phenotypes

Ploidy phenotypes

Refs.

SPOll-3 DNA topoisomerase VI

subunit A TOP6B DNA topoisomerase VI

subunit B RHL1 A component of DNA

topoisomerase VI ccs52 An activator of APC

HBT Subunit of APC

CYCA2;3 M cyclin CYCA2;1 M cyclin SIM ICK/KRP homolog

Developmental signalling

TRY MYB transcription factor

E3 ubiquitin ligase

RFI Not identified

UVI4/PYM Unknown protein

Light signalling

PHYB Photo receptor

Dwarf, reduced root hairs, small/under-branched trichomes5 Dwarf, reduced root hairs, small/under-branched trichomes5 Dwarf, reduced root hairs, small/under-branched trichomes5 Small petioles, hypocotyls, and roots6 Cell division arrest, increased cell size5 Not known5 Not known5

Multicellular trichomes5

Large and over-branched trichomes5

Large and over-branched trichomes, long hypocotyls5 Large and over-branched trichomes5 Large and over-branched trichomes, resistant to UV-B5

Long hypocotyls5

Reduced in hypocotyls, leaves, and trichomes Reduced in hypocotyls, leaves, and trichomes Reduced in hypocotyls, leaves, and trichomes Reduced in petioles, hypocotyls, and roots Increased in leaves

Increased in leaves Increased in seedlings Reduced in trichomes and hypocotyls

Increased in trichomes

Increased in trichomes and hypocotyls Increased in trichomes Increased in trichomes and hypocotyls

Increased in light-grown hypocotyls

Sugimoto-Shirasu et al. 2002; Härtung et al. 2002 Sugimoto-Shirasu et al. 2002; Härtung et al. 2002 Sugimoto-Shirasu et al. 2005

Cebolla et al. 1999

Serralbo et al. 2006

Imai et al. 2006 Yoshizumi et al. 2006 Walker et al. 2000; Churchman et al. 2006

Perazza et al. 1999; Schellmann et al. 2002 Perazza et al. 1999; El Refy et al. 2003 Perazza et al. 1999 Perazza et al. 1999; Hase et al. 2006

Gendreau et al. 1998

Gene

Expected function

General phenotypes

Ploidy phenotypes

Refs.

COP1

E3 ubiquitin ligase

DNA repair signalling

BRU1/TSKJ TPR + LRR protein

MG03

FAS1 Subunit of chromatin assembly factor

FAS2 Subunit of chromatin assembly factor

MSI1 Subunit of chromatin assembly factor

Plant hormone signalling

SPY O-GlcNAc transferase

CTR1 Raf family of MAPKKK

GA1 Ent-kaurene synthase

(GA-biosynthetic enzyme)

CPM17 P450

FRL1 Sterol methyltransferase

Constitutive photomorphogenesis5

Disorganized meristem, hypersensitive to DNA damage5 Disorganized meristem, increased cell size, hypersensitive to DNA damage5

Disorganized meristem, increased cell size, hypersensitive to DNA damage5

Disorganized meristem, increased cell size, hypersensitive to DNA damage6

Large and over-branched trichomes5

Ethylene triple response5 Short hypocotyls5

Short hypocotyls, brassinoride deficient5 Dwarf, abnormal petal shape, increased petal cell size5

Reduced in dark-grown hypocotyls

Increased in seedlings and flowers Increased in leaves and trichomes

Increased in leaves and trichomes

Increased in leaves and trichomes

Increased in leaves and trichomes Increased in hypocotyls Reduced in hypocotyls

Reduced in dark-grown hypocotyls Increased in petals

Gendreau et al. 1998

Takeda et al. 2004; Suzuki et al. 2005 Kaya et al 2001; Exner et al. 2006; Kirik et al. 2006 Kaya et al 2001; Exner et al. 2006

Kaya et al 2001; Exner et al. 2006

Jacobsen et al. 1996; Perazza et al. 1999 Gendreau et al. 1999 Gendreau et al. 1999

Gendreau et al. 1998

Hase et al. 2000, 2005

1 Phenotypes observed by 35S over-expression, 2 pGL2 expression, 3 activation tagging and 35S over-expression, 4 RNA interference, 5 EMS

mutation or T-DNA/tranposon insertion, 6 antisense interacts with DIMERIZATION PARTNER (DP) protein to activate the transcription of S-phase genes. There are three typical and three atypical E2Fs in Arabidopsis (Mariconti et al. 2002; Kosugi and Ohashi 2002). Co-expression of E2Fa, one of the typical E2Fs, and its interacting protein DPa, under 35S promoter promotes both cell division and endoreduplication (De Veylder et al. 2002), suggesting that E2Fa and DPa positively regulate both the mitotic cell cycle and endocycle (Fig. 3). In contrast, E2Fc, which also belongs to the typical E2F family (del Pozo et al. 2002), appears to promote the endocycle but inhibit the mitotic cell cycle since down-regulation of E2Fc expression by RNA interference leads to the formation of small and twisted leaves that have many small cells with reduced ploidy (del Pozo et al. 2006, Fig. 3). E2Fe/DP-E2F-like protein 1 (DEL1), one of the atypical E2Fs in Arabidopsis (Kosugi and Ohashi 2002), on the other hand, represses the endocycle since the del1-1 mutation leads to increased ploidy and the ectopic expression of E2Fe/DEL1 reduces endoreduplication (Vlieghe et al. 2005, Fig. 3). All of these Arabidopsis E2Fs can bind to the consensus cis-element to which authentic E2F bind (Mariconti et al. 2002), and the promotion or repression of the endocycle in these mutants and transgenic plants correlate with the altered expression of E2F target genes required for DNA replication (Vlieghe et al. 2005). DNA replication is licensed to occur when a pre-replication complex, composed of the origin recognition complex (ORC), CELL DIVISION CYCLE6 (CDC6), cyclin10 target 1 (CDT1) and the minichromosome maintenance (MCM) complex proteins, assembles at the replication origins. The transcription of these genes is up-regulated in E2Fa-DPa co-expressing lines (De Veylder et al. 2002), and even over-expression of CDC6 (Castellano et al. 2001) or CDT1 (Castellano et al. 2004) alone can drive extra cell division and the endocycle. Interestingly, promotion of these two different cell cycles is cell-type specific, i.e. cells that possess meristematic competence undergo further proliferation and cells that are committed to endoreduplication such as trichomes undergo another round of endocycle (Castellano et al. 2004), suggesting that the activity of the pre-replication complex is required for the progression of the endocycle but it is not involved in the mechanism that switches from the mitotic cell cycle to the endocycle (Fig. 3). Recent genetic studies show that the DNA topoiso-merase VI (topo VI) complex has a specific role in endoreduplication (Fig. 3). Arabidopsis mutants in the components of the plant topo VI complex such as HYPOCOTYL 6 (HYP6), ROOT HAIRLESS 2 (RHL2) and RHL1 (Sugimoto-Shirasu et al. 2002; Hartung et al. 2002; Sugimoto-Shirasu et al. 2005) are all viable and when they are induced to proliferate in callus induction media, they are capable of undergoing cell division at a similar rate to wild type. However, these mutants cannot complete the endocycle to reach 32C and instead they stall at 8C (Sugimoto-Shirasu et al. 2002, 2005; Hartung et al. 2002; Fig. 4). The plant topo VI complex is not likely to have a regulatory role in the endocycle. Instead, it probably acts during the S-phase in the endocycle to prevent the entanglement of replicated chromosomes.

Dwarf Mutant Arabidopsis

Fig. 4 Arabidopsis mutants that display ploidy phenotypes help us identify molecular components required for the endocycle. A A light micrograph of 2-week-old, light-grown wild-type (Col) and DNA topoisomerase VI mutant (top6) plants. The top6 mutants display extreme dwarf phenotypes. Scale 2 mm. B A light micrograph of 4-day-old, dark-grown wild-type (Col) and top6 hypocotyls. The growth of top6 hypocotyls is severely compromised. White triangles indicate the top and bottom of hypocotyls. Scale 4 mm. C A light micrograph of wild-type (Col) and top6 mutant roots. top6 is defective in the initiation and subsequent outgrowth of root hairs. Scale 1 mm. D Scanning electron micrographs of wild-type and top6 leaf epidermis demonstrate that the final cell size is reduced in top6. Scale 100 |im. E Flow cytometric analysis shows the ploidy of wild-type leaves ranges from 2C to 32C whereas the ploidy in top6 reaches only 8C, indicating that top6 has defects in the progression of successive endocycles beyond 8C

Fig. 4 Arabidopsis mutants that display ploidy phenotypes help us identify molecular components required for the endocycle. A A light micrograph of 2-week-old, light-grown wild-type (Col) and DNA topoisomerase VI mutant (top6) plants. The top6 mutants display extreme dwarf phenotypes. Scale 2 mm. B A light micrograph of 4-day-old, dark-grown wild-type (Col) and top6 hypocotyls. The growth of top6 hypocotyls is severely compromised. White triangles indicate the top and bottom of hypocotyls. Scale 4 mm. C A light micrograph of wild-type (Col) and top6 mutant roots. top6 is defective in the initiation and subsequent outgrowth of root hairs. Scale 1 mm. D Scanning electron micrographs of wild-type and top6 leaf epidermis demonstrate that the final cell size is reduced in top6. Scale 100 |im. E Flow cytometric analysis shows the ploidy of wild-type leaves ranges from 2C to 32C whereas the ploidy in top6 reaches only 8C, indicating that top6 has defects in the progression of successive endocycles beyond 8C

Another key process for cells to enter the endocycle is to drop the M-phase-specific CDK activity to inhibit the induction of mitosis. This process is primarily mediated through the down-regulation of M-phase-specific cy-clins, i.e. A-type and B-type cyclins (CYCA, CYCB, respectively), and several upstream regulators of these cyclins have been recently characterized (Fig. 3). The ilp1-1D mutant was originally identified as an increased ploidy mutant from Arabidopsis activation tagging lines (Yoshizumi et al. 2006). Various phenotypes including thick hypocotyls, large cotyledons, and long roots are observed at the early seedling stage in ilp1-1D, although its adult plants do not exhibit any obvious morphological change. ilp1-1D undergoes an extra round of endocycle and this phenotype is associated with the down-regulation of CYCA2 genes including CYCA2;1 and CYCA2;3, suggesting that ILP1 negatively regulates the expression of these CYCA2 genes (Fig. 3). Loss of function of CYCA2;1 and CYCA2;3 also induces an extra round of endocycle in young seedlings (Yoshizumi et al. 2006) and mature leaves (Imai et al. 2006), respectively. CYCA2;3 physically interacts with CDKA;1 and CDKA;1 is expressed in developed trichomes (Imai et al. 2006). Therefore, the CYCA2/CDKA;1 complex appears to act as a break for endoreduplication and increasing its activity is sufficient to terminate the endocycle (Fig. 3).

Another well-documented mechanism that allows down-regulation of M-phase CDK activity is the activation of the anaphase-promoting complex (APC), a ubiquitin ligase that targets CYCA and CYCB for degradation (Fig. 3). HOBITT (HBT1) is a subunit of the plant APC complex, and loss of HBT1 in hbt2311 inhibits cell proliferation and promotes the endocycle (Serralbo et al. 2006). Furthermore, a study of the legume Medicago trunc-tula has demonstrated that the APC complex is activated by its up-stream regulator CCS52/FIZZY-RELATED (FZR) protein, because suppression of the CCS52/FZR gene by the antisense gene causes reduced ploidy levels and small cells (Cebolla et al. 1999; Fig. 3). In Arabidopsis CYCA2;3 is one of the APC targets since over-expression of the stable CYCA2;3 that has a mutation in the destruction box strongly reduces ploidy level (Imai et al. 2006; Fig. 3).

M-phase-specific CDK activity is also regulated by inhibitors of CYC/CDK complexes such as ICK1/KRP1 and ICK2/KRP2 (Verkest et al. 2005, Weinl et al. 2005). High expression of these inhibitors blocks both the mitotic cell cycle and endocycle in Arabidopsis but their moderate expression appears to interfere only with the activity of mitotic CDKA, thus leading to an early entry into the endocycle (Verkest et al. 2005; Weinl et al. 2005). Another putative CDK inhibitor encoded by SIAMESE (SIM) interacts with D-type cyclin and CDKA;1 (Churchman et al. 2006). Loss of SIM function results in multicellular trichomes with individual cells having reduced ploidy (Walker et al. 2000), and this is associated with the ectopic expression of CYCB1;1 in trichomes (Churchman et al. 2006), suggesting that SIM inhibits the mitotic cell cycle in trichomes by down-regulating the expression of CYCB1. The WEE1 kinase, a negative regulator of CDK activity, is also suggested to have a role at the transition from the mitotic cell cycle to endocycle since its transcription is upregulated at the onset of endoreduplication in maize endosperm (Sun 1999; Fig. 3).

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