Endoreduplication in higher plants - Springer Link

Plant Molecular Biology 43: 735–745, 2000. Dirk Inzé (Ed.), The Plant Cell Cycle. © 2000 Kluwer Academic Publishers. Printed in the Netherlands.

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Endoreduplication in higher plants Jérôme Joubès and Christian Chevalier∗ Unité de Physiologie Végétale, Centre de Recherche INRA-Bordeaux, BP 81, 33883 Villenave d’Ornon Cedex, France (∗ author for correspondence; e-mail: [email protected])

Key words: cell cycle, development, endoreduplication

Abstract Cell polyploidisation can be achieved by endoreduplication, which consists of one or several rounds of DNA synthesis in the absence of mitosis. As a consequence, chromosomes with 2n chromatids are produced without change in the chromosome number. Endoreduplication is the most common mode of polyploidisation in plants and can be found in many cell types, especially in those undergoing differentiation and expansion. Although accumulating data reveal that this process is developmentally regulated, it is still poorly understood in plants. At the molecular level, the increasing knowledge on plant cell cycle regulators allows the acquisition of new tools and clues to understand the basis of endoreduplication control and, in particular, the switch between cell proliferation and cell differentiation.

Introduction The variation of cell ploidy levels designated as somatic polyploidy is a developmental process that has been described in many eukaryotes ranging from insects to mammals and plants (Brodsky and Uryvaeva, 1977). Polyploidisation results from the ability of cells to modify their classical cell cycle, in which DNA synthesis occurs independently from mitosis. This partial cell cycle is called the endoreduplication cycle or endocycle and accounts for the cessation of cell division and the increase in ploidy level. This phenomenon represents a growing field of interest in plant biology since it characterises the switch between cell proliferation and cell differentiation during developmental steps. Generally speaking, the classical cell cycle involves the accurate duplication and segregation of the chromosomal DNA stock, the subsequent cell division leading to the transmission of the genetic information from one mother cell to two daughter cells. Four distinct phases compose the cell cycle: a DNA presynthetic phase with a 2C nuclear DNA content (where C is the DNA content of the haploid genome), the G1 phase; the S phase during which DNA is synthesised,

with a nuclear DNA content intermediate between 2C and 4C; a DNA post-synthetic phase with a 4C nuclear DNA content, the G2 phase; and the M phase or mitosis. In the past ten years, increasing interest in the regulatory steps of the cell cycle has led to the characterisation of many cell cycle regulators such as the CDKs (cyclin-dependent kinases), the cyclins and different regulatory elements involved in the control of the progression throughout the cell cycle stages (Renaudin et al., 1996; Mironov et al., 1999). Knowledge of these plant regulatory elements and the mechanisms of cell cycle regulation will contribute to a better understanding of the endoreduplication cycle and its regulation. Cell polyploidy: endomitosis versus endoreduplication The designation ‘cell polyploidy’ is used as a general term to define the result of a multiple doubling (2n ) of nuclear DNA. Cell polyploidisation in both animals and plants is mainly due to either endomitosis or endoreduplication (D’Amato, 1964a, b; Brodsky and Uryvaeva, 1977). Other processes such as abortive mitosis, nuclei fusion or appearance of multinuclear

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Figure 1. Analysis of endoreduplication during tomato fruit development by flow cytometry. Tomato fruits were harvested at anthesis, 2, 5, 10, 15, 20 days after anthesis (DPA), mature green (MG), and red-ripe (RR) stages. Nuclei were then purified. From 10 DPA to RR stage, fruits were dissected into epidermis, pericarp and gel tissue prior to nuclei purification. In young developing fruits (up to 5 DPA) and in the epidermis even at the latest stage of development, nuclei display predominantly 2C and 4C DNA levels corresponding to the G1 and G2 phases respectively, thus indicating the dividing state of the tissues. In nuclei isolated from the pericarp and gel tissue, additional peaks of higher DNA content appear throughout development, from 10 DPA to RR stage. The appearance of such endoreduplication DNA profiles is concomitant with the start of the fruit growing period mainly by cell expansion (around 10 DPA). As the mitotic activity disappears in the gel after 10 DPA, a strong correlation is observed between endoreduplication and cell expansion leading to large and hypervacuolarized cells composing the gel tissue (Joubès et al., 1999).

cells can lead to polyploidisation, but are limited to ephemeral tissues (for a review, see D’Amato, 1984). Endomitosis, first described by Geitler (1939), occurs within an intact nuclear membrane and leads to a doubling of chromosome number during each endomitotic cycle. During endomitosis, chromosomes double and condense, and sister chromatids may separate and return to the interphase state as in the mitotic cycle. However, all of these events take place within the nuclear envelope and, as a result, nuclear endopolyploidy arises. Endomitosis differs essentially from mitosis by the absence of a mitotic spindle. It occurs in several

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animal groups but rarely in angiosperms (D’Amato, 1984). Endoreduplication was first described as a polyploid mitosis within the elongation zone of onion roots subjected to hormone treatment (Levan, 1939). It is the most common mode of cell polyploidisation in plants and is estimated to occur in over 90% of angiosperms (D’Amato, 1984). This process is an endonuclear chromosome duplication which occurs in the absence of any obvious condensation and decondensation steps leading to the production of chromosomes with 2n chromatids without any change in chromosome number (Lorz, 1947; Levan and Hauschka,

737 1953). As a consequence, in extreme cases, ‘giant’ chromosomes called polytene chromosomes may be formed within the hypertrophying nuclei by successive cycles of DNA replication without segregation of sister chromatids. Polyteny is a well documented nuclear differentiation mechanism reported in larval and adult Diptera and angiosperm ovular nuclei (D’Amato, 1984). Endoreduplicating nuclei retain the capability of DNA replication without going through mitosis. In a recent review dealing with the control of DNA replication, Grafi (1998) defined three types of endoreduplication cycles in which the normal restriction of one DNA replication per cell cycle is altered: (1) a multiple initiation of DNA replication within a given S phase, (2) a re-occurring S phase and (3) repeated S and Gap phases excluding the M phase. While the mechanism of the endoreduplication cycle is beginning to be well established in the model system Drosophila (Follette et al., 1998; Weiss et al., 1998), both the role and the control of endoreduplication cycle in plants are poorly understood.

Endoreduplication in plants: a general feature for polyploidisation Endoreduplication has been described in many specific cell types that are highly specialised and unusually large, such as raphid crystal idioblasts in Vanilla (Kausch and Horner, 1984), root hairs in Elodea canadensis (Dosier and Riopel, 1978), suspensor cells in Phaseolus (Nagl, 1974) and basal cells of the hairs on Bryonia anthers (Barlow, 1975). The latter two types of cells represent examples of a source of polytene chromosomes (with DNA levels of 4096C and 256C respectively). Other studies indicate that endoreduplication occurs in other types of tissues, such as the endosperm of maize kernels (Kowles and Phillips, 1985, 1988), parenchyma in orchid seedlings (Alvarez and Sagawa, 1965; Alvarez, 1968), cotyledons in peanut (Dhillon and Miksche, 1982), root parenchyma (Marciniak and Bilecka, 1985) and cortex cells from various species (Olszewska, 1976; Olszewska and Kononowicz, 1979), leaf epidermal cells in Phaseolus (Kinoshita, 1991) and Arabidopsis thaliana (Melaragno et al., 1993). New technologies such as flow cytometry analysis have opened the way for easy and rapid determination of C levels from large numbers of nuclei (Galbraith, 1983). In Mesembryanthemum crystallinum

and A. thaliana, nuclei were isolated from various tissues at different stages of plant development (De Rocher et al., 1990; Galbraith et al., 1991). A pattern of systematic somatic polyploidy could be defined as organ-specific. Older tissues show higher levels of polyploidy than younger ones within the same plant. In Cucumis sativus, polyploidy was described as a continuous process during successive developmental stages from seed to flowering plants (Gilissen et al., 1993). Polysomatic tissues were also found in species with large genomes such as Pisum sativum, Zea mays, Solanum tuberosum and Lycopersicon esculentum (Evans and van ’t Hof, 1975; Sree Ramulu and Dijkhuis, 1986; Biradar et al., 1993; Smulders, 1994). In tomato plants, polysomaty was found in all organs at all stages of development, with a typical distribution of a low level of polyploid nuclei in the small young organs, and high levels in fully differentiated organs such as ageing leaves (Smulders et al., 1994). In growing tomato fruits, endoreduplication occurs in all tissues but the epidermis (Bergervoet et al., 1996; Joubès et al., 1999) (Figure 1). The increase in nuclear DNA content resulting from endoreduplication is concomitant with the onset of cell expansion along with the arrest in mitotic activity. This observation is patent for the placental locular tissue composed of large and hypervacuolarised cells (Joubès et al., 1999) and is very similar to what was described for the developing endosperm of maize kernels (Kowles and Phillips, 1985; Grafi and Larkins, 1995). Nevertheless, direct evidence for the link between development and the degree of polyploidy in various organs and tissues of plants still remains to be found unequivocally.

Developmental, genetic, hormonal and environmental control of endoreduplication Epidermal hair cells called trichomes represent a good genetic system in which to analyse endoreduplication control during a developmental programme. In Arabidopsis, during morphogenesis, trichomes undergo four endoreduplication rounds leading to the formation of a large-branched single cell with a DNA content of 32C (Hülskamp et al., 1994, 1999). Trichome development has been studied extensively with Arabidopsis mutants (Martin and Glover, 1998; Traas et al., 1998) and is known to require many genes (Perazza et al., 1999). Some of these genes encode transcription factors that directly control the number

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738 of endocycles during trichome development. Furthermore, a direct correlation between the ploidy level and the number of branches is observed (Perazza et al., 1999). Hence endoreduplication associated with cell differentiation is thought to be genetically and developmentally regulated during trichome development (Traas et al., 1998). Plant hormones have been shown to affect cell division and cell elongation (Kende and Zeevaart, 1997). They may play an important role in the regulation of the endoreduplication process and in the control of the switch between the classical cell cycle and endoreduplication. Hormonal treatments of cultured cells are able to modify the status of dividing cells and induce extra rounds of DNA replication. In cultured pea root cortex cells, the presence of auxin and cytokinins induced an increase of nuclear DNA values to multiples of the 2C level with predominating 8C and 16C levels, as a result of doubling the chromosome number by endomitosis (Libbenga and Torrey, 1973). No change in DNA values was observed with auxin alone. These results suggested that cytokinins in the presence of auxin induced rounds of DNA synthesis prior to the first mitosis. In fact, this effect seems to be specific to the root cortex cells of leguminous plants as opposite results have been observed in different plant tissues. When auxin was applied to apricot trees, it caused a marked increase in fruit size due to the enlargement of mesocarp cell volume (Bradley and Crane, 1955). In mesocarp cells, the size of the nuclei is also increased consequently to the appearance of polyploidy. The hormone concentration used in the treatment was probably insufficient to stimulate mitosis in the mesocarp parenchyma but adequate to induce chromosome duplication by endomitosis (Bradley and Crane, 1955). Interestingly, this result suggests a possible relationship between the hormone gradient in fruit and the transition from mitosis to endopolyploidisation. Similarly, in cultured haploid Petunia leaf tissues, auxin treatment alone induces endopolyploidy by doubling the chromosome number (Liscum and Hangarter, 1991). The examples of hormonal regulation mentioned above deal with the induction of DNA replication but within an endomitosis context. The hormonal regulation of endoreduplication per se remains a relatively poorly documented area. Recently, it was shown that in cultured tobacco cells, an auxin-only signal induces elongation and DNA endoreduplication whereas in the presence of auxin and cytokinins cells divide actively

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(Valente et al., 1998). At the molecular level, cytokinins induce the rapid activation of the key cell cycle regulator CDKA through tyrosine dephosphorylation which is required for entry into mitosis (Zhang et al., 1996). This led these authors to propose the occurrence of a hormonal point control at the G2 phase stringently requiring cytokinins for progression into the M phase. However in an in planta system, RiouKhamlichi et al. (1999; p. 1543) demonstrated very clearly that the activation of the Arabidopsis cell cycle by cytokinins is exerted at the G1 -S boundary through the transcriptional regulation of D-type cyclin CycD3, thus appearing as potential mediators of plant mitogenic signals. Gibberellins have been shown to control DNA synthesis during hypocotyl development in Arabidopsis (Gendreau et al., 1999). The use of the same Arabidopsis GA-deficient mutants has shown that gibberellins could induce trichome development through up-regulation of genes required for trichome initiation (Perraza et al., 1999). Using uniconazol (a GA biosynthesis inhibitor), Perraza et al. (1998) showed that gibberellins can induce both trichome number and branch number during Arabidopsis growth. Gibberellins promote trichome formation through induction of GL1 (GLABROUS1) expression. Gendreau et al. (1999, p. 516) suggested that gibberellic acid has a stimulating effect on the overall process of plant development rather than a direct effect on endoreduplication. This supports the previous observation of the absence of effect of gibberellic acid on the percentage distribution of C values in pea epicotyls (Callebaut et al., 1982). Ethylene has been shown to induce an extra round of endoreduplication during Arabidopsis hypocotyl elongation (Gendreau et al., 1999). However, like gibberellic acid, this effect seems to be much more a consequence of multiple responses to the hormone rather than a direct effect on endoreduplication. Plant growth conditions seem to influence polyploidy patterns. Leaves of tomato plants grown in a greenhouse show higher levels of polyploidy than leaves of in vitro grown tomato plants (Smulders et al., 1994). The same variability has been also observed for potato plants (Uijtewaal, 1987). The growth condition effect could be related to environmental factors such as the light regime. During elongation in the dark of the epicotyl of Pisum sativum and the hypocotyl of Arabidopsis thaliana, an endocycle appears leading to 8C and 16C DNA levels respectively, while the nuclear DNA levels in the light are 4C and 8C (van Oosteveldt and van Parijs, 1975; Gendreau et al.,

739 1997). Furthermore, Gendreau et al. (1998, p. 226) showed that inhibition of this third endocycle in lightgrown Arabidopsis hypocotyls is not the consequence of a simple feed-back mechanism coupling the number of cycles to the cell volume, but an integral part of the phytochrome-controlled photomorphogenic programme. In Arabidopsis as well as in tomato, potato and maize (Biradar et al., 1993; Pijnacker et al., 1989; Smulders et al., 1994; Gendreau et al., 1998), diploid and tetraploid plants grown in the same environmental conditions display similar patterns of polyploidy in the corresponding organs, suggesting that the developmental and environmental programme determining the number of endoreduplication rounds could be independent of the initial nuclear DNA content.

Molecular mechanisms of the plant endoreduplication cycle In the past decade, our knowledge of the cell cycle has developed considerably towards the understanding of how the progression within the different stages of the cell cycle is regulated. Assuming the endoreduplication cycle is a modified cell cycle, it must share common determinants with the classical cell cycle. Especially with regard to the control of the G1 /S transition, recent data have opened the way towards the understanding of the regulation of endoreduplication in plants. In eukaryotes, a common class of heterodimeric protein complexes regulates the progression within the cell cycle. These complexes consist of a catalytic subunit referred to as CDK and a regulatory cyclin subunit. The activity of the complexes is governed by phosphorylation/dephosphorylation events, availability of the protein partners (synthesis and degradation of the cyclin moiety) and binding to other proteins such as inhibitors or activators (Lees, 1995). Based on multiple sequence alignments between the 30 or so CDKs identified so far in plants (Burssens et al., 1998; Mironov et al., 1999), a nomenclature has been proposed defining at least five distinct classes of CDKs (Joubès et al., this issue). The most numerous class, CDKA, groups functional homologues of the yeast p34cdc2/CDC28 protein which are characterised by the presence of the PSTAIRE motif essential for cyclin binding (Ducommun et al., 1991). CDKA appears to be constitutively expressed throughout the cell cycle (Segers et al., 1997), and is in fact associated with the competence of the cells to divide (Hemerly

et al., 1993). CDKB proteins present unique features which indicate that these kinases may represent examples of mitotic kinases with putative plant-specific functions for entry into or progression through the M phase (Burssens et al., 1998). Hence both CDKA and CDKB are associated with dividing cells and mitotic activity. CDKC, CDKD and CDKE form less numerous classes for which very little functional information is available. In situ hybridisation performed on vegetative shoot apices of Arabidopsis thaliana showed that CDKB transcripts are only expressed in dividing tissues (Jacqmard et al., 1999). In endoreduplicating tissues, CDKB gene expression is abolished while CDKA transcripts remain slightly detectable (Jacqmard et al., 1999). At the translational level, it was shown that the CDKA protein level is unaffected and consequently constitutively expressed, in the endoreduplicating maize endosperm and locular tissue of tomato fruit (Grafi and Larkins, 1995; Joubès et al., 1999). Most interestingly, it was demonstrated in these two developmental systems that the H1 kinase activity specific of M-phase CDKs is highly reduced during the endoreduplication process. Hence these data suggest that endoreduplication can affect both the expression of CDKA and CDKB at the transcriptional level (Jacqmard et al., 1999), and at the post-translational level (Grafi and Larkins, 1995; Joubès et al., 1999). The following post-translational mechanisms, namely CDK phosphorylation/dephosphorylation, lack or degradation of the corresponding mitotic cyclin, and inactivation by a CDK inhibitor, may account for the decrease in the M-phase CDK histone H1 kinase activity. The use of chemical drugs such as staurosporine known to inhibit protein kinases was shown to induce endoreduplication in mammalian and plant cells. The treatment of rat diploid fibroblasts with the staurosporine analogue K252a resulted in DNA re-replication (Usui et al., 1991). In cell suspension cultures of Phaseolus, K252a treatment induced continuous cell cycles without mitosis and led to the appearance of polyploidy levels of up to 2048C (Nagl, 1993). Furthermore, K252a was found to inhibit p34cdc2 and p34cdc2-like kinases (Gadbois et al., 1992). During the development of maize endosperm which is characterised by the inhibition of mitosis and subsequent endoreduplication, Sun et al. (1999a, p. 4184) showed that a cDNA coding for the maize homologue of the Wee1 kinase is up-regulated suggesting that its potential phosphorylation activity on CDKA Thr-14 and Tyr-15 residues influences CDK

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740 activity through a strong post-translational inhibitory mechanism. Altogether, these results obtained from different models suggest that inhibitory phosphorylation of mitotic CDKs is involved in the control of endoreduplication. An outstanding report described the isolation and the functional analysis of a mitotic inhibitor named CCS52. For the first time was demonstrated the involvement of a cell cycle regulator linking cell proliferation to cell differentiation and promoting endoreduplication and cell enlargement during root nodule formation in Medicago sativa (Cebolla et al., 1999). CCS52 was identified as being a WD-repeat regulatory protein of the fizzy-related family, an activator of the anaphase-promoting factor (APC) which is involved in the ubiquitin-dependent proteolysis of mitotic cyclins and consequently in the inactivation of M-phase CDK kinase activity (King et al., 1995). When overexpressed in yeast, ccs52 triggered cell division arrest through the degradation of mitotic cyclin which was followed by endoreduplication and cell enlargement. The expression of ccs52 is associated in planta with differentiating and endoreduplicating cells, and partial impairment by an antisense gene results in a decrease in nuclear polyploidy and a consequent decrease in cell size. The unavailability of mitotic cyclins triggering M-phase CDK inactivation is thus controlled at the post-translational level by proteolytic degradation. Yet, the transcriptional regulation of mitotic cyclin gene expression is also an important determinant during endoreduplication. In maturing leaves of Arabidopsis (Jacqmard et al., 1999), in the course of maize endosperm development (Sun et al., 1999b) and tomato fruit development (Joubès et al., 2000), the down-regulation of mitotic Arath;CycB1;1, Zeama;CycB1;3 and tomato mitotic cyclins Lyces;CycA1;1, Lyces;CycA2;1, Lyces;CycB1;1 and Lyces;CycB2;1 was demonstrated during the transition from mitosis to endoreduplication. The presence of a proteinaceous CDK inhibitor might also be a determinant of cell division arrest in endoreduplicating tissues by interacting with the M-phase CDKs. In yeast, Moreno and Nurse (1994, p. 236) identified the rum1+ (replication uncoupled mitosis) gene, which encodes an inhibitor of the mitotic CDK. Overexpression of rum1+ in fission yeast cells induces endoreduplication and nucleus enlargement. In plants, the involvement of such an inhibitor is suspected from the following experiments. An inhibitory protein from endoreduplicating tissues can suppress M-phase activity in maize endosperm (Grafi

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and Larkins, 1995). Similarly, in synchronised alfalfa cells, Bögre et al. (1997, p. 849) showed that an inhibitory protein inactivates CDK activity isolated from S-phase extracts. The first two reported examples of such CDK inhibitors have been recently characterised in Arabidopsis (Wang et al., 1997; Lui et al., 2000). These inhibitors named ICK1 and ICK2 were shown to interact only with Arath;CDKA (Wang et al., 1998; Lui et al., 2000). ICK1 and ICK2 transcripts display distinct expression patterns with the highest level in mature leaves and stems respectively, i.e. in endoreduplicating tissues (Galbraith et al., 1991). Whether these CKIs are truly involved in the impairment of M-phase CDK activity during endoreduplication still remains to be addressed. The CKS protein belongs to the putative CDKinteracting proteins. It is thought to function as a docking factor on CDK proteins for both positive and negative regulators of kinase activity. CKS1At, the Arabidopsis homologue of p13Suc1/Cks1 protein, binds both Arath;CDKA1 and Arath;CDKB1 in vitro and in vivo (De Veylder et al., 1997). Jacqmard et al. (1999, p. 496) investigated the role of CKS1At during endoreduplication. These authors analysed its putative role in the process of endoreduplication by in situ hybridisation experiments on the vegetative shoot apices of Arabidopsis plants. The expression of CKS1At was observed in both mitotic and endoreduplicating tissues, indicating a role for CKS1At in both type of cycles. As the expression of Arath;CDKA1 and Arath;CDKB1 was almost restricted to mitotically active cells, it was then hypothesised that CKS1At might be necessary for the regulation of an as yet uncharacterised CDK putatively involved in the entry and progression throughout the S phase of both cycles. The implication of other types of CDK in the endoreduplication process is supported by the fact that the onset of endoreduplication in maize endosperm is concomitant with the activation of S phase-related protein kinases (Grafi and Larkins, 1995). These complexes display the ability to phosphorylate the maize retinoblastoma protein (Rb) (Grafi et al., 1996) known to be implicated with D-type cyclins in the checkpoint control of the progression from G1 to S phase (for a review see Gutierrez et al., 1998, and de Jager and Murray, 1999). According to the mammalian model, Rb and its homologues sequester transcription factors such as E2F. The phosphorylation of Rb by CDK/cyclin complexes results in the release of the transcription factor E2F and the induction of S-phase gene transcription. The putative S-phase-related pro-

741 tein kinases from maize endosperm display the ability to interact with human E2F and the adenovirus E1A proteins (Grafi and Larkins, 1995). During the development of maize endosperm, changes in the level and state of Rb phosphorylation coincide with the initiation of endoreduplication (Grafi et al., 1996). In the same manner, it was shown that Rb-immunoreactive 110 kDa proteins accumulate in the differentiated zones of maize leaves, whereas they are barely detectable in the basal zone (Huntley et al., 1998). At the transcriptional level, the newly isolated NtRb1 cDNA encoding the tobacco homologue for Rb was shown to be highly expressed in differentiated tissues and poorly detectable in undifferentiated BY-2 cells in suspension culture (Nakagami et al., 1999). Maize Rb was shown to be phosphorylated by purified human CDK/cyclin complexes such as CDK2/cycA and the G1 /S transition-specific CDK4/cycD and CDK2/cycE complexes (Huntley et al., 1998). However, in plants, no homologue counterparts of the mammalian G1 kinases CDK4/6 associated with the D-type cyclins has been described so far. Thus the identification of plant G1 /S-specific kinases, as well as their subunit composition (types of CDK and cyclin partner) are still speculative. Recently, Nakagami et al. (1999, p. 247) demonstrated that the tobacco Rb-related protein was phosphorylated in vitro by a CDKA/CycD complex. This result raises the question of whether the CDKA/CycD complex plays a role in G1 /S transition, and may represent a G1 -specific kinase complex. Nevertheless, it still remains to be demonstrated that this unusual complex is active in phosphorylating the Rb protein in vivo. Among the distinct classes of plant cyclins defined according to sequence similarities with animal homologues, only the mitotic cyclins of the A and B types and the G1 cyclins of D type have been identified (Renaudin et al., 1996). No homologue for cyclin E has been found so far in plants. In animals and especially in Drosophila, cyclin E is known to play an important role in the control of the endoreduplication cycle. Indeed, during endoreduplication, the expression of mitotic cyclins is abolished whereas cyclin E expression is transiently increased (Follette et al., 1998; Weiss et al., 1998). The CDK2/cycE activity which triggers DNA replication needs to be downregulated to allow a subsequent S phase in vivo. As mentioned above, in plant endoreduplicating tissues the transcripts for mitotic cyclins become undetectable as they are no longer expressed (Jacqmard et al., 1999; Sun et al., 1999b; Joubès et al., 2000). In the en-

doreduplicating locular tissues of developing tomato fruits (Figure 1; Joubès et al., 1999), Lyces;CycD3;1 is induced concomitantly with the increase of nuclear ploidy level (Joubès et al., 2000), and interestingly, the maximum expression of Lyces;CycA3;1 is obtained when the ploidy level is the highest. In the light of these results, it is tempting to speculate that D-type cyclins (at least Lyces;CycD3;1) and A3-type cyclins (e.g. Lyces;CycA3;1) play a crucial role, in the G-to-S phase transition and in the progression through the S phase of the plant endoreduplication cycle respectively. Integrating our current knowledge emerging from the few molecular data presented herein, we propose a still partial model for the endoreduplication cycle control (Figure 2). In this model, we integrated the different ways of inactivating the mitotic CDK/cyclin complexes which were demonstrated to occur during endoreduplication using various plant developmental systems. The CDK moiety of the G-to-S CDK/cyclin complex remains to be identified, or confirmed as being CDKA as suggested by recent works from Nakagami et al. (1999, p. 246). Further investigations dealing with the basic mechanisms that regulate the cell cycle are necessary to provide information and tools which will help to complete this model and understand the molecular control of endoreduplication.

Conclusion: what is the need for endoreduplication? Endoreduplication is the major mechanism leading to somatic polyploidisation in plants. Evident correlations have been established between polyploidy and cell differentiation and cell expansion. However the intriguing question which remains to be answered concerns the real physiological role of endoreduplication. It has been proposed that somatic polyploidy may represent an evolutionary strategy which substitutes for a lack of phylogenetic increase in nuclear DNA (Nagl, 1976). Endopolyploidy appears to be most prevalent in plants harboring small genomes. The development of systemic polyploidy may reflect the requirement of certain specialised cells for a minimal mass of nuclear DNA in order to maintain specific regulatory and functional states (Nagl, 1976; Galbraith et al., 1991). Because it occurs in tissues displaying high metabolic activity such as silk glands in dipterans or endosperm of developing maize kernels, it has also been suggested that endoreduplication may provide a

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Figure 2. Schematic representation of endoreduplication cycle control in plants. Taking into account recent progress dealing with endoreduplication and the cell cycle control, a model for the endocycle regulation in plants is proposed. During development, endoreduplication occurs in the absence of mitosis. The arrest of the mitotic activity in endoreduplicating tissues may originate from the inactivation of the mitotic CDK/cyclin complexes (CDKA/B / CycA/B). This inactivation may be achieved by fixation of cyclin-dependent kinase inhibitor (CKI), modification of the CDK phosphorylation status by the Wee1 kinase or lack of the A- and B-type mitotic cyclins resulting from the down-regulation of their transcription or their degradation by the APC/26S proteasome pathway. The activation of the APC may be mediated by specific transcription factors such as CCS52. During the endocycle, D-type cyclins (CycD) are produced and associate with a still uncharacterized CDK (CDK?) resulting in the formation of complexes able to phosphorylate the retinoblastoma protein (Rb). The G-to-S transition occurs due to the release of the E2F transcription factor, which is necessary for the transcription of S-phase-related genes, in particular the A-type cyclins (CycAs ). PEST motifs in the primary sequence trigger the rapid degradation of CycD. The induced CycAs at the onset of the S-phase associate with CDK? forming the complexes regulating the progression throughout the S phase. At the end of the S phase, CycAs are degraded by the APC/26S proteasome pathway; CDK? is then released and available for a new round of the endoreduplication cycle.

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743 mechanism whereby cells increase the availability of DNA template in order to increase the level of gene expression. However, the direct correlation between the increase in DNA template and an improved yield of transcription and subsequent translation still is to be demonstrated. With regard to the notion of a defined mass of nuclear DNA in relation to cellular functions, Galbraith et al. (1991) invoked the coordination of gene expression, which is required for the correct interaction of nuclear and organellar genomes. During plant development, endoreduplication could accommodate the enhanced transcription of nuclear genes needed for the assembly of organelles with the increase in the numbers of organelles and organellar genomes per cell. A body of evidence suggests that endoreduplication is tightly linked to increases in nuclear volume and/or cell size. Genetical analyses emphasised the involvement of endoreduplication in plant developmental processes such as cell expansion and cell differentiation (Traas et al., 1998). It has been proposed that endoreduplication could be considered as a mechanism to generate sufficient DNA in anticipation of a future massive increase in tissue mass. Rather than strictly determining the final size of cells, specific endoreduplication levels would contribute to set a range of cell volumes to be reached according to the response to environmental factors (Traas et al., 1998). In relation with the emergence of this range of cell volumes, endoreduplication may also favour cell size diversity by allowing selective (non-synchronous) cell division arrest. Even if endoreduplication and cell expansion are tightly correlated during development, the determinants of this coupling have not yet been elucidated. Likewise, the relationship, either causal or consequential, between endopolyploidy and differentiation during development is not known. Endoreduplication results from an altered cell cycle, the endocycle, in which mitosis is absent. Most of the key cell cycle molecular factors such as CDKs, cyclins, proteolytic machinery elements, putative activators and inhibitors, are also part of the endocycle. Therefore, the apparent difference between the classical cell cycle and the endocycle may reside in the differential regulation of the expression of these components, at least at the transcriptional or posttranslational level. In a more general context, a better understanding of the molecular mechanisms related to polyploidy and endoreduplication in plants could be useful for

improving yield and quality of plant products, as endoreduplication seems to be of paramount importance in increasing the biomass of agronomic species (Nagl, 1978).

Acknowledgements We would like to thank Dr P. Raymond and Dr B. Ricard (Unité de Physiologie Végétale, INRABordeaux) for critically reading the manuscript. This work was supported by grant 95-5-23722 from the Ministère de la Recherche et de la Technologie (France) to J.J.

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