FZR2/CCS52A1 Expression Is a Determinant of Endoreduplication and Cell Expansion in Arabidopsis

Endoreduplication, a modified cell cycle that allows cells to increase ploidy without subsequent cell division, is a key component of plant growth and development. In this work, we show that some, but not all, of the endoreduplication of Arabidopsis (Arabidopsis thaliana) is mediated by the expression of a WD40 gene, FIZZY-RELATED2 (FZR2). Loss-of-function alleles show reduced endoreduplication and reduced expansion in trichomes and other leaf cells. Misexpression of FZR2 is sufficient to drive ectopic or extra endoreduplication in leaves, roots, and flowers, leading to alteration of cell sizes and, sometimes, organ size and shape. Our data, which suggest that reduced cell size can be compensated by increased cell proliferation to allow normal leaf morphology, are discussed with respect to the so-called compensation mechanism of plant development.A key element of plant morphogenesis is the balance among cell proliferation, expansion, and differentiation to produce organs of the characteristic sizes and shapes. At the heart of plant morphogenesis is the cell versus organism debate. On one hand, the cell theory postulates that the cells of a multicellular organism behave autonomously, and the sum of their activities results in the morphology of that organism (Schwann, 1839). On the other hand, the organismal theory postulates that morphology is governed by a genetic mechanism separate from the behavior of individual cells (Kaplan and Hagemann, 1991). Regardless of perspective, it is clear that the plant program of cell growth and proliferation is dynamic and responsive, i.e. perturbations in one factor can be compensated by modifications in the other (Day and Lawrence, 2000; Mizukami, 2001). For example, forcing a decrease in leaf cell number by overexpressing the cell cycle regulator KRP2 results in an increase in cell volume (De Veylder et al., 2001). Conversely, an increase in cell volume caused by the overexpression of ABP1 leads to a decrease in cell number (Jones et al., 1998). This compensation mechanism has been incorporated into a hybrid cell/organismal theory called “neo-cell theory” (Tsukaya, 2003). The nature of this compensation mechanism is unclear, although it likely involves cellular reaction to organ-level positional information.During the development of many plants, certain cell types undergo extensive endoreduplication, a modified cell cycle that results in DNA replication without subsequent mitosis. In leaves of Arabidopsis (Arabidopsis thaliana), several rounds of endoreduplication occur in trichomes and in most epidermal pavement cells, while guard cells remain diploid (Melaragno et al., 1993). In the last decade, extensive research has looked at the trichome as a model to unravel the mechanism controlling endoreduplication. During trichome development, four rounds of endoreduplication regularly take place, although variation exists (Melaragno et al., 1993). Some Arabidopsis mutants show concomitant changes in trichome ploidy and branch number, supporting a role of endoreduplication in the control of branch number (Hülskamp et al., 1994; Perazza et al., 1999; Downes et al., 2003). Branching changes are not always tied to endoploidy changes, however; for example, plants with loss-of-function alleles or overexpression of STICHEL show trichome branch number alterations without changes in nuclear DNA content (Ilgenfritz et al., 2003). Endoreduplication appears to be an important determinant of cell and organ size. Indeed, a correlation between cell size and endoploidy has been found in Arabidopsis and other plant species (Galbraith et al., 1991; Traas et al., 1998; Kudo and Kimura, 2002), although Arabidopsis root cells constitute an exception (Beemster et al., 2002), and certain genetic manipulations can also uncouple this relationship (Hemerly et al., 1993; Wang, et al., 2000; De Veylder et al., 2001; Fujikura et al., 2007). Interestingly, different levels of endoreduplication may affect the same tissue in distinct species. For instance, orchid (Oncidium varicosum and Phalaenopsis spp.) and cabbage (Brassica capitata) petals are large and undergo extensive endoreduplication, with some cells reaching 64C (Kudo and Kimura, 2001; Lee et al., 2004). The sizes of petal cells, and hence of petal organs, directly correlate with their ploidy in these studies. By contrast, the cone cells of Arabidopsis petals remain diploid despite being capable of endoreduplication (Hase et al., 2005), and this plant shows relatively small flowers in comparison to close relatives. Unlike orchid and cabbage, Arabidopsis is self-fertilizing, so producing small petals would not hinder its reproduction.Similar cellular components regulate endoreduplication and the mitotic cell cycle. Cyclin-dependent kinases (CDKs) control the cell cycle by forming complexes with cyclins, by phosphorylation or dephosphorylation, or by association with CDK inhibitors. Expression and degradation of specific cyclins promote progression through distinct stages of the cell cycle and control the exit from the cell cycle. For example, D-type cyclins become phosphorylated and associate with the A-type CDK to induce the switch from G1 phase to S phase (Inzé and De Veylder, 2006). Destruction of B1-type cyclins seems to be necessary for completion of the M phase, because introduction of a B1 cyclin for which the destruction-box motif has been altered can prevent cytokinesis (Weingartner et al., 2004). In yeast and Drosophila, Fizzy-Related (FZR) family proteins have been shown to trigger the degradation of A- and B-type cyclins by targeting them to the Anaphase Promoting Complex/Cyclosome (APC/C), an E3 ubiquitin protein ligase (Sigrist and Lehner, 1997; Yamaguchi et al., 1997). In those studies, misexpression of FZR family proteins was able to induce endoreduplication. Furthermore, a recent study linked the APC/C to local cell expansion, endoreduplication, and the compensation mechanism in Arabidopsis (Serralbo et al., 2006).The plant homologs of FZR were first implicated in endoreduplication in root nodules of Medicago sativa, and antisense expression of the Medicago truncatula Cell-cycle Switch 52 A and B (Ccs52A and Ccs52B), orthologs of FZR, led to reduced endoreduplication in that species (Cebolla et al., 1999). In Arabidopsis, three FZR homologs exist, and in cell cultures, FZR1 and FZR2 showed similar high expression in G1 and S phases of the cell cycle, while FZR3 expression increased at the end of G2 and beginning of M. All three FZR proteins associated to free and CDK-bound A- and B-type cyclins (Fülöp et al., 2005). Although the FZR genes are known to be expressed in all major tissues (Beemster et al., 2005), the normal developmental function of the FZR family has not yet been determined in Arabidopsis.In this study, we examined the developmental roles of one of the Arabidopsis FZR homologs, FZR2. We found that FZR2 expression is necessary for correct cell expansion and endoreduplication, and its misexpression is sufficient to induce extra or ectopic endoreduplication and cell expansion. We gathered empirical data that address the putative compensation mechanism balancing cell proliferation with cell expansion.     

Potential role of the rice OsCCS52A gene in endoreduplication

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

DNA topoisomerase VI is essential for endoreduplication in Arabidopsis

Endoreduplication is a common process in eukaryotes that involves DNA amplification without corresponding cell divisions. Cell size in various organisms has been linked to endoreduplication, but the molecular mechanisms are poorly understood. We have used a genetic strategy to identify molecules involved in endocycles in Arabidopsis. We isolated two extreme dwarf mutants, hypocotyl6 (hyp6) and root hairless2 (rhl2) [3], and cells of these mutants successfully complete only the first two rounds of endoreduplication and stall at 8C. In both mutants, large cell types, such as trichomes and some epidermal cells, that normally endoreduplicate their DNA are much reduced in size. We show that HYP6 encodes AtTOP6B, a plant homolog of the archaeal DNA topoisomerase VI subunit B, and that RHL2 encodes AtSPO11-3, one of the three Arabidopsis subunit A homologs. We propose that this topoisomerase VI complex is essential for the decatenation of replicated chromosomes during endocycles and that successive rounds of endoreduplication are required for the full growth of specific cell types.     

Signaling role of fructose mediated by FINS1/FBP in Arabidopsis thaliana

Sugars are evolutionarily conserved signaling molecules that regulate the growth and development of both unicellular and multicellular organisms. As sugar-producing photosynthetic organisms, plants utilize glucose as one of their major signaling molecules. However, the details of other sugar signaling molecules and their regulatory factors have remained elusive, due to the complexity of the metabolite and hormone interactions that control physiological and developmental programs in plants. We combined information from a gain-of-function cell-based screen and a loss-of-function reverse-genetic analysis to demonstrate that fructose acts as a signaling molecule in Arabidopsis thaliana. Fructose signaling induced seedling developmental arrest and interacted with plant stress hormone signaling in a manner similar to that of glucose. For fructose signaling responses, the plant glucose sensor HEXOKINASE1 (HXK1) was dispensable, while FRUCTOSE INSENSITIVE1 (FINS1), a putative FRUCTOSE-1,6-BISPHOSPHATASE, played a crucial role. Interestingly, FINS1 function in fructose signaling appeared to be independent of its catalytic activity in sugar metabolism. Genetic analysis further indicated that FINS1-dependent fructose signaling may act downstream of the abscisic acid pathway, in spite of the fact that HXK1-dependent glucose signaling works upstream of hormone synthesis. Our findings revealed that multiple layers of controls by fructose, glucose, and abscisic acid finely tune the plant autotrophic transition and modulate early seedling establishment after seed germination.     

New insights into the control of endoreduplication: endoreduplication could be driven by organ growth in Arabidopsis leaves

Enormous progress has been achieved understanding the molecular mechanisms regulating endoreduplication. By contrast, how this process is coordinated with the cell cycle or cell expansion and contributes to overall growth in multicellular systems remains unclear. A holistic approach was used here to give insight into the functional links between endoreduplication, cell division, cell expansion, and whole growth in the Arabidopsis (Arabidopsis thaliana) leaf. Correlative analyses, quantitative genetics, and structural equation modeling were applied to a large data set issued from the multiscale phenotyping of 200 genotypes, including both genetically modified lines and recombinant inbred lines. All results support the conclusion that endoreduplication in leaf cells could be controlled by leaf growth itself. More generally, leaf growth could act as a "hub" that drives cell division, cell expansion, and endoreduplication in parallel. In many cases, this strategy allows compensations that stabilize leaf area even when one of the underlying cellular processes is limiting.     

DDB2, DDB1A and DET1 exhibit complex interactions during Arabidopsis development

Damaged DNA-binding proteins 1 and 2 (DDB1 and DDB2) are subunits of the damaged DNA-binding protein complex (DDB). DDB1 is also found in the same complex as DE-ETIOLATED 1 (DET1), a negative regulator of light-mediated responses in plants. Arabidopsis has two DDB1 homologs, DDB1A and DDB1B. ddb1a single mutants have no visible phenotype while ddb1b mutants are lethal. We have identified a partial loss-of-function allele of DDB2. To understand the genetic interaction among DDB2, DDB1A, and DET1 during Arabidopsis light signaling, we generated single, double, and triple mutants. det1 ddb2 partially enhances the short hypocotyl and suppresses the high anthocyanin content of dark-grown det1 and suppresses the low chlorophyll content, early flowering time (days), and small rosette diameter of light-grown det1. No significant differences were observed between det1 ddb1a and det1 ddb1a ddb2 in rosette diameter, dark hypocotyl length, and anthocyanin content, suggesting that these are DDB1A-dependent phenotypes. In contrast, det1 ddb1a ddb2 showed higher chlorophyll content and later flowering time than det1 ddb1a, indicating that these are DDB1A-independent phenotypes. We propose that the DDB1A-dependent phenotypes indicate a competition between DDB2- and DET1-containing complexes for available DDB1A, while, for DDB1A-independent phenotypes, DDB1B is able to fulfill this role.     

Gibberellin and ethylene control endoreduplication levels in the Arabidopsis thaliana hypocotyl

We have previously shown that endoreduplication levels in hypocotyls of Arabidopsis thaliana (L.) Heynh. are under negative control of phytochromes. In this study, the hormonal regulation of this process was analysed using a collection of A. thaliana mutants. The results show that two hormones in particular, gibberellin (GA) and ethylene, play distinct roles. Hypocotyl cells of the GA-deficient mutant ga1-11 grown in the dark did not elongate and showed a greatly reduced endoreduplication. Normal endoreduplication could be restored by supplying 10⁻⁹ M of the gibberellin GA₄₊₇, whereas the restoration of normal cell growth required 100-fold higher concentrations. The GA-insensitive mutant gai showed reduced cell elongation but normal ploidy levels. We conclude that (i) GA₄₊₇ has a global positive effect on endoreduplication and (ii) that endoreduplication is more sensitive to GA₄₊₇ than cell elongation. Ethylene had a completely different effect. It induced an extra round of endoreduplication both in light- and dark-grown seedlings and acted mainly on discrete steps rather than having a global effect on endoreduplication. The genes EIN2 and CTR1, components of the ethylene signal transduction pathway were both involved in this process. Received: 27 February 1999 / Accepted: 21 May 1999     

The immunophilin-interacting protein AtFIP37 from Arabidopsis is essential for plant development and is involved in trichome endoreduplication

The FKBP12 (FK506-binding protein 12 kD) immunophilin interacts with several protein partners in mammals and is a physiological regulator of the cell cycle. In Arabidopsis, only one specific partner of AtFKBP12, namely AtFIP37 (FKBP12 interacting protein 37 kD), has been identified but its function in plant development is not known. We present here the functional analysis of AtFIP37 in Arabidopsis. Knockout mutants of AtFIP37 show an embryo-lethal phenotype that is caused by a strong delay in endosperm development and embryo arrest. AtFIP37 promoter::beta-glucuronidase reporter gene constructs show that the gene is expressed during embryogenesis and throughout plant development, in undifferentiating cells such as meristem or embryonic cells as well as highly differentiating cells such as trichomes. A translational fusion with the enhanced yellow fluorescent protein indicates that AtFIP37 is a nuclear protein localized in multiple subnuclear foci that show a speckled distribution pattern. Overexpression of AtFIP37 in transgenic lines induces the formation of large trichome cells with up to six branches. These large trichomes have a DNA content up to 256C, implying that these cells have undergone extra rounds of endoreduplication. Altogether, these data show that AtFIP37 is critical for life in Arabidopsis and implies a role for AtFIP37 in the regulation of the cell cycle as shown for FKBP12 and TOR (target of rapamycin) in mammals.     

A RING-H2 zinc-finger protein gene RIE1 is essential for seed development in Arabidopsis

RING zinc-finger proteins play important roles in the regulation of development in a variety of organisms. In the plant kingdom, few genes encoding RING zinc-finger proteins have been documented with visible effects on plant growth and development. A novel gene, RIE1, encoding a RING-H2 zinc-finger protein was identified in Arabidopsis thaliana and is characterized in this paper. RIE1 encodes a predicted protein product of 359 amino acids residues with a molecular mass of 40 kDa, with a RING-H2 zinc-finger motif located at the extreme end of the C-terminus. Characterization of a Dissociation (Ds) insertion line (SGT4559) and a T-DNA insertion line (SRIE1) demonstrated that disruption of RIE1 is embryo-lethal. SGT4559 heterozygous plants produced seeds with embryo development arrested from globular to torpedo stages. Some mutant seeds were rescued by embryo culture, and the mutant (rie1) plants seemed to grow normally compared to wild-type plants, except that the mutants produced only abnormal seeds. However, RIE1 was expressed in different tissues throughout the whole plant as revealed by northern blot analysis and gene fusion assay of RIE1 promoter with the beta-glucuronidase (GUS) gene. Our results indicated that RIE1 plays an essential role in seed development.     

Misexpression of the cyclin-dependent kinase inhibitor ICK1/KRP1 in single-celled Arabidopsis trichomes reduces endoreduplication and cell size and induces cell death

A positive correlation between cell size and DNA content has been recognized in many plant cell types. Conversely, misexpression of a dominant-negative cyclin-dependent kinase (CDK) or CDK inhibitor proteins (ICK/KRPs) in Arabidopsis and tobacco leaves has revealed that cell growth can be uncoupled from cell cycle progression and DNA content. However, cell growth also appears to be controlled in a non-cell-autonomous manner by organ size, making it difficult in a ubiquitous expression assay to judge the cell-autonomous function of putative cell growth regulators. Here, we investigated the function of the CDK inhibitor ICK1/KRP1 on cell growth and differentiation independent of any compensatory influence of an organ context using Arabidopsis trichomes as a model system. By analyzing cell size with respect to DNA content, we dissected cell growth in a DNA-dependent and a DNA-independent process. We further found that ICK1/KRP1 misexpression interfered with differentiation and induced cell death, linking cell cycle progression, differentiation, and cell death in plants. The function of ICK1/KRP1 in planta was found to be dependent on a C-terminal domain and regulated negatively by an N-terminal domain. Finally, we identified CDKA;1 and a D-type cyclin as possible targets of ICK1/KRP1 expression in vivo.     

Ectopic endoreduplication caused by sterol alteration results in serrated petals in Arabidopsis

The Arabidopsis frill1 (frl1) mutant, that has serrated petals and sepals but no other large changes in plant morphology, was studied. The frl1 had a mutation in STEROL METHYLTRANSFERASE 2 and an altered sterol composition. It was found that the frl1 mutation causes ectopic endoreduplication in petal tips that do not normally endoreduplicate. The rosette leaves of frl1 also showed an enhanced level of endoreduplication, but their morphology was hardly affected. These facts suggest that the suppression of endoreduplication is important for petal morphogenesis and the normal sterol composition is required for this suppression.     

CRM1/BIG-mediated auxin action regulates Arabidopsis inflorescence development

The shape of the inflorescence in Arabidopsis thaliana ecotype Columbia is a raceme with individual flowers developing acropetally. The ecotype Landsberg harboring the erecta (er) mutation shows a corymb-like inflorescence, namely a compact inflorescence with a flattened arrangement of flower buds at the tip. To gain insight into inflorescence development, we previously isolated corymb-like inflorescence mutants, named corymbosa1 (crm1), and found that the corymb-like inflorescence in crm1-1 was due to reduced cell elongation of pedicels and stem internodes. Double mutants of crm1 with er and crm2, and crm1-1 crm2-1 er-105 triple mutants show an additive phenotype. crm1-1 is caused by a mutation in BIG, which is required for polar auxin transport. CRM1/BIG is expressed in inflorescence meristems, floral meristems and vascular tissues. We analyzed a collection of 12 reduced lateral root formation (rlr) mutants, which are allelic to crm1-1, and categorized the mutants into three classes, depending on the plant developmental defects. Although all 12 alleles had new stop codons, the phenotype of heterozygous crm1-1/doc1-1 and Northern blotting suggest that new crm1/big mutant alleles are hypomorphic. Auxin-responsive DR5rev::GFP expression was decreased in crm1-1 vasculature of pedicels and stem internodes. PINFORMED1 (PIN1) and CRM1/BIG are expressed in vasculature of pedicels and stem internodes. The severity of corymb-like inflorescence in crm1/big mutants correlated with increased levels of PIN1. Our results suggest that CRM1/BIG controls the elongation of the pedicels and stem internodes through auxin action.     

Phytochrome controls the number of endoreduplication cycles in the Arabidopsis thaliana hypocotyl

A majority of the cells in the Arabidopsis hypocotyl undergo endoreduplication. The number of endocycles in this organ is partially controlled by light. Up to two cycles occur in light-grown hypocotyls, whereas in the dark about 30% of the cells go through a third cycle. Is the inhibition of the third endocycle in the light an indirect result of the reduced cell size in the light-grown hypocotyl, or is it under independent light control? To address this question, the authors examined the temporal and spacial patterns of endoreduplication in light- or dark-grown plants and report here on the following observations: (i) during germination two endocycles take place prior to any significant cell expansion; (ii) in the dark the third cycle is completed very early during cell growth; and (iii) a mutation that dramatically reduces cell size does not interfere with the third endocycle. The authors then used mutants to study the way light controls the third endocycle and found that the third endocycle is completely suppressed in far red light through the action of phytochrome A and, to a lesser extent, in red light by phytochrome B. Furthermore, no 16C nuclei were observed in dark-grown constitutive photomorphogenic 1 seedlings. And, finally the hypocotyl of the cryptochrome mutant, hy4, grown in blue light was about three times longer than that of the wild-type without a significant difference in ploidy levels. Together, the results support the view that the inhibition of the third endocycle in light-grown 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 program.     

The A-type cyclin CYCA2;3 is a key regulator of ploidy levels in Arabidopsis endoreduplication

Plant cells frequently undergo endoreduplication, a process in which chromosomal DNA is successively duplicated in the absence of mitosis. It has been proposed that endoreduplication is regulated at its entry by mitotic cyclin-dependent kinase activity. However, the regulatory mechanisms for its termination remain unclear, although plants tightly control the ploidy level in each cell type. In the process of searching for regulatory factors of endoreduplication, the promoter of an Arabidopsis thaliana cyclin A gene, CYCA2;3, was revealed to be active in developing trichomes during the termination period of endoreduplication as well as in proliferating tissues. Taking advantage of the situation that plants encode highly redundant cyclin A genes, we were able to perform functional dissection of CYCA2;3 using null mutant alleles. Null mutations of CYCA2;3 semidominantly promoted endocycles and increased the ploidy levels achieved in mature organs, but they did not significantly affect the proportion of cells that underwent endoreduplication. Consistent with this result, expression of the CYCA2;3-green fluorescent protein fusion protein restrained endocycles in a dose-dependent manner. Moreover, a mutation in the destruction box of CYCA2;3 stabilized the fusion protein in the nuclei and enhanced the restraint. We conclude that CYCA2;3 negatively regulates endocycles and acts as a key regulator of ploidy levels in Arabidopsis endoreduplication.